Production of organic acids from aspergillus cis-aconitic acid decarboxylase (cada) deletion strains

ABSTRACT

This application provides recombinant  Aspergillus  fungi having an endogenous cis-aconitic acid decarboxylase (cadA) gene genetically inactivated, which allows aconitic acid production by the recombinant fungi. Such recombinant fungi can further include an exogenous nucleic acid molecule encoding aspartate decarboxylase (panD), an exogenous nucleic acid molecule encoding β-alanine-pyruvate aminotransferase (BAPAT), and an exogenous nucleic acid molecule encoding 3-hydroxypropironate dehydrogenase (HPDH). Kits including these fungi, and methods of using these fungi to produce aconitic acid and 3-hydroxypropionic acid (3-HP) are also provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 16/393,149filed Apr. 24, 2019, which claims priority to U.S. ProvisionalApplication No. 62/661,804 filed Apr. 24, 2018, both herein incorporatedby reference in their entireties.

ACKNOWLEDGMENT OF GOVERNMENT SUPPORT

This disclosure was made with Government support under ContractDE-AC05-76RL0 1830 awarded by the U.S. Department of Energy. TheGovernment has certain rights in the invention.

FIELD

Recombinant Aspergillus genetically inactivated for an endogenouscis-aconitic acid decarboxylase (cadA) gene are provided, as are methodsof using such recombinant fungi to produce aconitic acid and3-hydroxypropionic acid (3-HP).

BACKGROUND

Itaconic acid (IA) is utilized as a monomer or co-monomer to formpolymers that are used as raw material for plastics, resins, syntheticfibers and elastomers, detergents and cleaners. Aspergillus terreusThom, produces an appreciable amount of itaconic acid when grown in aglucose medium. Cell-free extracts of Aspergillus terreus containcis-aconitic decarboxylase (cadA), which can decarboxylate cis-aconiticacid into equal moles of itaconic acid and carbon dioxide.

The itaconic acid gene cluster (IA cluster) includes four genes,including cis-aconitic acid decarboxylase (cadA), a predictedtranscription factor (tf), mitochondrial organic acid transporter(mttA), and MFS (Major Facilitator Superfamily) type transporter (mfsA)located in plasma membranes. Expression of one or more genes of the IAgene cluster in hetereologous hosts, including E. coli, A. niger, and S.cerevisiae, can result in the production of itaconic acid in nonitaconic acid host microorganisms.

Characterization and regulation of genes in the IA biosynthesis clusterthrough gene deletion had not been previously investigated. Theinventors used protoplast transformation to delete each gene in the IAcluster in Aspergillus terreus/Aspergillus pseudoterreus, which allowedfor the effect on cell growth and IA production to be investigated.

SUMMARY

The role of cis-aconitic acid decarboxylase (cadA), a predictedtranscription factor (tf), mitochondrial organic acid transporter(mttA), and MFS (Major Facilitator Superfamily) type transporter (mfsA)in IA biosynthesis in A. pseudoterreus ATCC 32359 is shown herein.Expressed Sequence Tag (EST) analysis showed a similar expressionpattern among those four genes distinct from neighboring genes.Systematic gene deletion analysis demonstrated that tf, cadA, mttA andmfsA genes in the cluster are essential for IA production.Interestingly, significant amounts of aconitic acid production wasdetected in the cadA deletion strain but not in the other deletionstrains.

Based on these observations, a novel recombinant ΔcadA Aspergillusstrain is provided, which can be used for aconitic acid and otherorganic acid production. Provided herein are isolated recombinant fungi(such as Aspergillus filamentous fungi) having a gene inactivation (alsoreferred to herein as a gene deletion or functional deletion) of acis-aconitic acid decarboxylase (cadA) gene (referred to herein as AcadAstrains). In some examples, the Aspergillus fungi is Aspergillus terreusor Aspergillus pseudoterreus, or particular strains thereof (for exampleA. pseudoterreus ATCC32359 and A. terreus NRRL 1960). In particularexamples, a ΔcadA strain exhibits one or more of the followingcharacteristics: produces at least 2-fold, at least 3-fold,at least 3.5fold, at least 5-fold, at least 8-fold, or at least 10-fold more totalaconitic acid than a wild-type Aspergillus terreus or Aspergilluspseudoterreus (for example at day 3, 4, 5, 6, 7, 8, 9 or 10 ofproduction); produces at least 2-fold more cis-aconitic acid at day 5,6, 7, 8, 9, or 10 of culturing in Riscaldati medium than a wild-typeAspergillus terreus or Aspergillus pseudoterreus; produces at least2-fold, at least 3-fold, at least 5-fold, or at least 10- fold moretrans-aconitic acid at day 10 of culturing in Riscaldati medium than awild-type Aspergillus terreus or Aspergillus pseudoterreus; orcombinations thereof. In some examples, such increases are relative toAspergillus terreus strain ATCC 32359 grown under the same conditions.

In particular examples, a ΔcadA fungi further includes an exogenousnucleic acid molecule encoding aspartate 1-decarboxylase (panD), anexogenous nucleic acid molecule encoding β-alanine-pyruvateaminotransferase (BAPAT), and an exogenous nucleic acid moleculeencoding 3-hydroxypropionate dehydrogenase (HPDH). The ΔcadA fungiexpressing panD, BAPAT, and HPDH can be used to produce 3-HP. Suchexogenous nucleic acid molecules can be part of one or more exogenousnucleic acid molecules, such as 1, 2 or 3 exogenous nucleic acidmolecules. In one example, the exogenous nucleic acid molecule encodingpanD has at least 80%, at least 90%, at least 95%, at least 98%, or 100%sequence identity to SEQ ID NO: 53 or 65 and/or encodes a panD proteinhaving at least 80%, at least 90%, at least 95%, at least 98%, or 100%sequence identity to SEQ ID NO: 54. In one example, the exogenousnucleic acid molecule encoding BAPAT has at least 80% , at least 90%, atleast 95%, at least 98%, or 100% sequence identity to SEQ ID NO: 55,and/or encodes a BAPAT protein having at least 80%, at least 90%, atleast 95%, at least 98%, or 100% sequence identity to SEQ ID NO: 56. Inone example, the exogenous nucleic acid molecule encoding HPDH has atleast 80%, at least 90%, at least 95%, at least 98%, or 100% sequenceidentity to SEQ ID NO: 57, and/or encodes a HPDH protein having at least80%, at least 90%, at least 95%, at least 98%, or 100% sequence identityto SEQ ID NO: 58. Such panD, BAPAT, and HPDH nucleic acid molecules canbe part of a vector. In addition, expression of the panD, BAPAT, andHPDH can be driven by one or more promoters.

The endogenous cadA gene is genetically inactivated in some examples bya deletion mutation (complete or partial) or by insertional mutation(e.g., by insertion of an antibiotic resistance gene, such ashygromycin). In some examples, prior to its genetic inactivation, thecadA gene encodes a protein having at least 80%, at least 90%, at least95%, at least 98%, or 100% sequence identity to SEQ ID NO: 50 or 52. Insome examples, prior to its genetic inactivation, the cadA gene (or acadA coding sequence) comprises at least 80%, at least 90%, at least95%, at least 98%, or 100% sequence identity to SEQ ID NO: 49, 51, 59 or92.

Also provided herein are compositions (such as a culture media orfermentation broth) and kits that include a Aspergillus ΔcadA strain.Also provided herein are compositions (such as a culture media orfermentation broth) and kits that include an Aspergillus AcadA strainthat also express panD, BAPAT, and HPDH, in some examples such genes areexogenous to the fungi. In some examples, the composition or kitincludes Riscaldati medium (such as modified Riscaldati medium with 20×trace elements).

Also provided herein are methods of making aconitic acid (such ascis-aconitic acid, trans-aconitic acid, or both) using the disclosedAspergillus ΔcadA strains. For example, such a method can includeculturing an isolated ΔcadA Aspergillus under conditions that permit thefungus to make aconitic acid, thereby producing aconitic acid. Forexample, the AcadA fungus can be cultured in Riscaldati medium. In someexamples, the method further includes isolating the aconitic acidproduced, for example isolating it from the culture media or from thefungus.

Also provided herein are methods of making 3-hydroxypropionic acid (3-HPusing the disclosed Aspergillus ΔcadA strains that also expresses panD,BAPAT, and HPDH (which can be exogenous). For example, such a method caninclude culturing an isolated ΔcadA Aspergillus that also expressespanD, BAPAT, and HPDH under conditions that permit the fungus to make3-HP, thereby producing 3-HP. For example, the ΔcadA fungus that alsoexpresses panD, BAPAT, and HPDH can be cultured in Riscaldati medium(such as one including 20× trace elements). In some examples, the methodfurther includes isolating the 3-HP produced, for example isolating itfrom the culture media or from the fungus.

The foregoing and other objects and features of the disclosure willbecome more apparent from the following detailed description, whichproceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Hypothesized itaconic acid (IA) production and transport pathwayin Aspergillus pseudoterreus and Aspergillus terreus. Glucose isutilized by A. terreus and A. pseudoterreus to form pyruvate and issubsequently converted to citric acid for tricarboxylic acid (TCA) cyclein the mitochondria. Citric acid is dehydrated to cis-aconitic acid,which is then transported from mitochondria to cytosol throughtransporter mttA. In the cytosol, cis-aconitic acid is decarboxylatedinto itaconic acid and CO2 by cis-aconitic decarboxylase. Finally,itaconic acid secreted outside of cell through transporters, for examplemfsA.

FIG. 2. Aspergillus pseudoterreus ATCC 32359 fermentation data forcollecting samples for EST sequencing. A 20 L volume of Riscaldatiproduction medium (see Riscaldati et al., J Biotechnol 2000, 83:219-230)in a 30 L working volume Sartorius fermenter was inoculated with 10⁶ A.pseudoterreus spores per ml. The three samples referred to as“preproduction, production onset and production” were collected at 40,50 and 62 hours, respectively. Itaconic acid and glucose data are shownon the left y-axis and fungal ash free dry weight (AFDW) is shown on theright y-axis.

FIGS. 3A-3B. Aspergillus pseudoterreus IA Cluster Analysis after fivedays growth in Riscadati medium. Spores 0.5×10⁸ were inoculated into 50ml of production media for IA production as described in Riscaldati etal. (J Biotechnol 2000, 83:219-230). The cultivation was performed at30° C. on a rotary shaker at 150 rpm. At the end of five days, sampleswere obtained for HPLC analysis and biomass measurement. (A) Dry massmeasurement of wild type and mutant strains (B) Itaconic acid productionof wild type and mutant strains. The average obtained from threeindependent experiments are shown. Error bars represent standarddeviations from the means.

FIG. 4. Kinetics of itaconic acid production by wild type A.pseudoterreus and Δtf strains grown in production media at 30° C. Spores0.5×10⁸ were inoculated into 50 ml of production media for itaconic acidproduction as described in Riscaldati et al. (J Biotechnol 2000,83:219-230). The cultivation was performed at 30° C. on a rotary shakerat 150 rpm. All experiments were done in three replicates. At day 2, 4,6, and 7, HPLC analysis was performed to determine amount of IAproduced. Each sample was measured in five replicates. Error barsrepresent standard deviation from the means.

FIG. 5. Real-time (RT)-PCR analysis of the relative levels of mttA,cadA, mfsA mRNAs in wild type and Δtf strains. Spores 0.5×10⁸ wereinoculated into 50 ml of production media for itaconic acid productionas described in Riscaldati et al. (J Biotechnol 2000, 83:219-230). Thecultivation was performed at 30° C. on a rotary shaker at 150 rpm. Allexperiments were done in three biological replicates. At day 3, sampleswere collected and RNA was extracted for RT-PCR. The average of resultsobtained from five independent RNA preparations is shown. All transcriptlevels were measured in triplicate for each RNA preparation. Error barsrepresent standard deviations from the means. Compared to wild type,expression level of mttA, cadA and mfsA were decreased 57, 37 and 23fold in the Δtf strain.

FIGS. 6A-6C. Aconitic acid production in ΔcadA strain. The cultivationwas performed at 30° C. on a rotary shaker at 150 rpm. All experimentswere done in three biological replicates. (A) at day 5, only ΔcadAproduced cis-aconitic and trans-aconitic acid, while wild type and othermutants did not. (B) Time course of cis- and trans-aconitic acidproduction in Δcad strain over 10 days. (C) Comparison of total aconiticacid production between wild type and ΔcadA mutant strains.

FIG. 7. Arrangement of transgene expression cassette for 3-HP Productionin A. pseudoterreus with a synthetic beta-alanine pathway. A descriptionof each Fragment is described in Example 8. The relevant fragments werecloned into pBlueScript SK(−) vector linearized with restriction enzymeH3/PstI. The whole expression cassette was linearized with restrictionenzyme Xhol for the protoplast transformation for homologousrecombination at cadA locus.

FIG. 8. Southern blot confirmation of cadA gene interruption by 3HPtransgene expression cassette (FIG. 7). The cadA gene in the transgenicstrains #2 (3HP-2) and #6 (3HP-6) was disrupted by the homologousrecombination, while the random integration occurred in the strains #4(3HP-4) and #5 (3HP-5). No insertion was observed in strains #1 and #3.

FIGS. 9A-9B. 3-HP production. A. pseudoterreus having a geneticallyinactivated cadA locus alone (cad1Δ), or additionally expressing panD,BAPAT, and HPDH (3HP-2, 3HP-4, 3HP-5, and 3-HP6), were grown at 30° C.on a rotary shaker at 200 rpm for (A) 7 days, or (B) over 8 days, in theRiscaldati media with 20× TE, and 3-HP present in the supernatantmeasured using HPLC.

SEQUENCE LISTING

The nucleic acid sequences listed in the accompanying sequence listingare shown using standard abbreviations for nucleotide bases and aminoacids, as defined in 37 C.F.R. 1.822. Only one strand of each nucleicacid sequence is shown, but the complementary strand is understood asincluded by any reference to the displayed strand. The sequence listingsubmitted herewith, generated on Feb. 5, 2021, 80 kb, is hereinincorporated by reference. In the accompanying sequence listing:

SEQ ID NOS: 1-8 are primers used to delete the tf gene in A.pseudoterreus.

SEQ ID NOS: 9-16 are primers used to delete the mttA gene in A.pseudoterreus.

SEQ ID NOS: 17-24 are primers used to delete the cadA gene in A.pseudoterreus.

SEQ ID NOS: 25-32 are primers used to delete the mfsA gene in A.pseudoterreus.

SEQ ID NOS: 33-40 are primers used to delete the p450 gene in A.pseudoterreus.

SEQ ID NOS: 41-42 are primers used to amplify mttA in A. pseudoterreus.

SEQ ID NOS: 43-44 are primers used to amplify cadA in A. pseudoterreus.

SEQ ID NOS: 45-46 are primers used to amplify mfsA in A. pseudoterreus.

SEQ ID NOS: 47-48 are primers used to amplify benA in A. pseudoterreus.

SEQ ID NOS: 49 and 50 are exemplary cadA nucleic acid and proteinsequences, respectively, from A. terreus (GenBank Accession Nos.AB326105.1 and BAG49047.1).

SEQ ID NOS: 51 and 52 are exemplary cadA nucleic acid and proteinsequences, respectively, from A. vadensis CBS 113365 (GenBank AccessionNos. XM_025706777.1 and XP_025563141.1).

SEQ ID NOS: 53 and 54 are exemplary aspartate 1-decarboxylase (panD)nucleic acid and protein sequences, respectively, from Triboliumcastaneum (GenBank Accession Nos. NM_001102585.1 and NP_001096055.1).Coding sequence nt 41-1663.

SEQ ID NOS: 55 and 56 are exemplary β-alanine-pyruvate aminotransferase(BAPAT) nucleic acid and protein sequences, respectively, from Bacilluscereus AH1272 (GenBank Accession Nos. ACMS01000158.1 (complement (10606. . . 11961)) and EEL86940.1).

SEQ ID NOS: 57 and 58 are exemplary 3-hydroxypropionate dehydrogenase(HPDH) nucleic acid and protein sequences (GenBank Accession No.WP_000636571), respectively.

SEQ ID NO: 59 is an A. pseudoterreus 5′-cadA nucleic acid sequence.

SEQ ID NOS: 60-61 are primers used to isolate an A. pseudoterreus5′-cadA gene.

SEQ ID NO: 62 is an A. niger gpdA promoter nucleic acid sequence.

SEQ ID NOS: 63-64 are primers used to isolate an A. niger gpdA promoter.

SEQ ID NO: 65 is panD cDNA of Tribolium castaneum with codonoptimization for A. pseudoterreus.

SEQ ID NOS: 66-67 are primers used to isolate panD cDNA of Triboliumcastaneum with codon optimization for A. pseudoterreus.

SEQ ID NO: 68 is a bidirectional terminator from A. nigerelf3/multifunctional chaperone.

SEQ ID NOS: 69-70 are primers used to isolate bidirectional terminatorfrom A. niger elf3/multifunctional chaperone.

SEQ ID NO: 71 is codon optimized synthetic cDNA of β-alanine-pyruvateaminotransferase (BAPAT) of Bacillus cereus.

SEQ ID NOS: 72-73 are primers used to isolate a codon optimizedsynthetic cDNA of BAPAT of Bacillus cereus.

SEQ ID NO: 74 is an A. niger eno1 promoter.

SEQ ID NOS: 75-76 are primers used to isolate an A. niger eno1 promoter.

SEQ ID NO: 77 is an A. nidulans gpdA promoter.

SEQ ID NOS: 78-79 are primers used to isolate an A. nidulans gpdApromoter.

SEQ ID NO: 80 is the codon optimized synthetic cDNA of E. coli3-hydroxypropionate dehydrogenase (HPDH).

SEQ ID NOS: 81-82 are primers used to isolate a codon optimizedsynthetic cDNA of E. coli HPDH.

SEQ ID NO: 83 is a trpC terminator of A. nidulans.

SEQ ID NOS: 84-85 are primers used to isolate the trpC terminator of A.nidulans.

SEQ ID NO: 86 is a trpC terminator of A. nidulans.

SEQ ID NOS: 87-88 are primers used to isolate a trpC terminator of A.nidulans.

SEQ ID NO: 89 is an A. oryzae ptrA selection marker gene.

SEQ ID NOS: 90-91 are primers used to isolate the A. oryzae ptrAselection marker gene.

SEQ ID NO: 92 is an A. pseudoterreus 3′-cadA gene.

SEQ ID NOS: 93-94 are primers used to isolate an A. pseudoterreus3′-cadA gene fragment.

SEQ ID NO: 95 is a combination of Fragments 7 to 9 (SEQ ID NOS: 77, 80,and 83, respectively).

SEQ ID NO: 96 is a primer used to isolate Fragments 7 to 9 (incombination with SEQ ID NO: 88).

SEQ ID NO: 97 is a combination of Fragments 11 and 12 (SEQ ID NOS: 89and 92, respectively).

SEQ ID NO: 98 is a primer used to isolate Fragments 11 to 12 (incombination with SEQ ID NO: 90).

DETAILED DESCRIPTION

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this disclosure belongs. Definitions of commonterms in molecular biology may be found in Benjamin Lewin, Genes V,published by Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrewet al. (eds.), The Encyclopedia of Molecular Biology, published byBlackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers(ed.), Molecular Biology and Biotechnology: a Comprehensive DeskReference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8).

The singular terms “a,” “an,” and “the” include plural referents unlesscontext clearly indicates otherwise. Similarly, the word “or” isintended to include “and” unless the context clearly indicatesotherwise. Hence “comprising A or B” means including A, or B, or A andB. It is further to be understood that all base sizes or amino acidsizes, and all molecular weight or molecular mass values, given fornucleic acids or polypeptides are approximate, and are provided fordescription. Although methods and materials similar or equivalent tothose described herein can be used in the practice or testing of thepresent disclosure, suitable methods and materials are described below.All publications, references and Genbank® Accession numbers (thesequence available on Apr. 24, 2019) mentioned herein are incorporatedby reference in their entireties. The materials, methods, and examplesare illustrative only and not intended to be limiting.

In order to facilitate review of the various embodiments of thedisclosure, the following explanations of specific terms are provided:

3-hydroxypropionate dehydrogenase (HPDH): EC 1.1.1.59 An enzyme thatcatalyzes the chemical reaction:3-hydroxypropanoate+NAD^(±)⇄3-oxopropanoate+NADH+H⁺. The term HPDHincludes any HPDH gene (such as a bacterial or fungal panD sequence),cDNA, mRNA, or protein, that is a HPDH that can covert3-hydroxypropanoate and NAD⁺ into 3-oxopropanoate, NADH, and H⁺ and viceversa. Expression or increased expression of HPDH, for example in anAspergillus also expressing BAPAT and panD and having a geneticallyinactivated cadA gene (ΔcadA), results in a fungus that has an abilityto produce more 3-HP than the parent strain (such as at least 20%, atleast 30%, at least 40%, 50%, at least 60% at least 70%, at least 100%,at least 200%, at least 300%, or at least 400% more than a parent strainunder the same growing conditions).

HPDH sequences are publicly available. For example, SEQ ID NO: 57discloses an HPDH coding sequence and GenBank® Accession No:WP_000636571 discloses an HPDH protein sequence (SEQ ID NO: 58);GenBank® Accession Nos. FR729477.2 (nt 1005136 . . . 1005885) andCBY27203.1 disclose exemplary Yersinia enterocolitica subsp. palearcticaY11 HPDH nucleic acid and protein sequences, respectively; and GenBank®Accession Nos: CP004083.1 (complement(1399227 . . . 1399973) andAJQ99264.1 disclose exemplary Enterobacteriaceae bacterium bta3-1 HPDHnucleic acid and protein sequences, respectively. However, one skilledin the art will appreciate that in some examples, a HPDH sequence caninclude variant sequences (such as allelic variants and homologs) thatretain HPDH activity and when expressed in an Aspergillus alsoexpressing BAPAT and panD and with a genetically inactivated cadA gene(ΔcadA), results in a fungus that has an ability to produce more 3-HPthan the parent strain (such as at least 20%, at least 30%, at least40%, 50%, at least 60% at least 70%, at least 100%, at least 200%, atleast 300%, or at least 400% more than a parent strain under the samegrowing conditions).

Aconitic acid: An organic acid with two isomers, cis- and trans-aconiticacid. The ΔcadA fungi provided herein can be used to produce cis- andtrans-aconitic acid.

Aspartate 1-decarboxylase (panD): EC 4.1.1.11. An enzyme that catalyzesthe chemical reaction: L-aspartate⇄beta-alanine+CO₂. The term panDincludes any panD gene (such as a bacterial or fungal panD sequence),cDNA, mRNA, or protein, that is a panD that can covert L-aspartate intobeta-alanine+CO₂ and vice versa. Expression or increased expression ofpanD, for example in an Aspergillus also expressing BAPAT and HPDH andhaving a genetically inactivated cadA gene (ΔcadA), results in a fungusthat has an ability to produce more 3-HP than the parent strain (such asat least 20%, at least 30%, at least 40%, 50%, at least 60% at least70%, at least 100%, at least 200%, at least 300%, or at least 400% morethan a parent strain under the same growing conditions)

panD sequences are publicly available. For example, GenBank® AccessionNos: NM_001102585.1 and NP_001096055.1 disclose Tribolium castaneum panDnucleic acid and protein sequences, respectively (SEQ ID NOS: 55 and56); GenBank® Accession Nos. CP002745.1 (complement(4249351 . . .4249824)) and AEK63458.1 disclose exemplary Collimonas fungivoransTer331 panD nucleic acid and protein sequences, respectively; andGenBank® Accession Nos: CP029034.1 (nt 1201611 . . . 1201994) andAWE15802.1 disclose exemplary Bacillus velezensis panD nucleic acid andprotein sequences, respectively. However, one skilled in the art willappreciate that in some examples, a panD sequence can include variantsequences (such as allelic variants and homologs) that retain panDactivity and when expressed in an Aspergillus also expressing BAPAT andHPDH and with a genetically inactivated cadA gene (ΔcadA), results in afungus that has an ability to produce more 3-HP than the parent strain(such as at least 20%, at least 30%, at least 40%, 50%, at least 60% atleast 70%, at least 100%, at least 200%, at least 300%, or at least 400%more than a parent strain under the same growing conditions).

β-alanine-pyruvate aminotransferase (BAPAT): EC 2.6.1.18. An enzyme thatcan catalyze the reactionL-alanine+3-oxopropanoate⇄beta-alanine+pyruvate. The term BAPAT includesany BAPAT gene (such as a bacterial or fungal panD sequence), cDNA,mRNA, or protein, that is a BAPAT that can convert beta-alanine andpyuvate to L-alanine and 3-oxopropanoate [or malonic semialdehyde], andvice versa. Expression or increased expression of BAPAT, for example inan Aspergillus also expressing HPDH and panD and having a geneticallyinactivated cadA gene (ΔcadA), results in a fungus that has an abilityto produce more 3-HP than the parent strain (such as at least 20%, atleast 30%, at least 40%, 50%, at least 60% at least 70%, at least 100%,at least 200%, at least 300%, or at least 400% more than a parent strainunder the same growing conditions).

BAPAT sequences are publicly available. For example, GenBank® AccessionNos: ACMS01000158.1 (complement(10606 . . . 11961)) and EEL86940.1disclose Bacillus cereus AH1272 BAPAT nucleic acid and proteinsequences, respectively (SEQ ID NOS: 55 and 56); GenBank® Accession Nos.DF820429.1 (complement (241627 . . . 242967)) and GAK28710.1discloseexemplary Serratia liquefaciens FK01 BAPAT nucleic acid and proteinsequences, respectively; and GenBank Accession Nos: LGUJ01000001.1complement (92812 . . . 94140) and KOY12524.1 disclose exemplaryBradyrhizobium diazoefficiens BAPAT nucleic acid and protein sequences,respectively. However, one skilled in the art will appreciate that insome examples, a BAPAT sequence can include variant sequences (such asallelic variants and homologs) that retain BAPAT activity and whenexpressed in an Aspergillus also expressing HPDH and panD and with agenetically inactivated cadA gene (AcadA), results in a fungus that hasan ability to produce more 3-HP than the parent strain (such as at least20%, at least 30%, at least 40%, 50%, at least 60% at least 70%, atleast 100%, at least 200%, at least 300%, or at least 400% more than aparent strain under the same growing conditions).

cadA (cis-aconitic acid decarboxylase): The cadA gene encodes an enzyme(EC 4.1.1.6) that catalyzes the chemical reactioncis-aconitate⇄itaconate+CO₂. The term cadA (or cadA) includes any cadAgene (such as a fungal cadA sequence), cDNA, mRNA, or protein, that is acadA that can catalyze the decarboxylation of cis-aconitate to itaconateand CO₂ and vice versa, and when genetically inactivated results in afungus that produces more aconitic acid than the parent strain without agenetically inactivated cadA gene (such as at least 20%, at least 30%,at least 50%, at least 60%, at least 75%, at least 100%, at least 200%,at least 500%, or 1000% more than a parent strain under the same growingconditions, for example at day 5 of production). In some examples, aparental strain containing a functional native cadA sequence does notproduce detectable aconitic acid. In some examples, genetic inactivationof cadA results in a fungus that produces more trans-aconitic acid thancis-aconitic acid at day 10 of production, (such as at least 2-fold, atleast 3-fold, at least 4-fold, or at least 5-fold more at day 10 ofproduction).

cadA sequences are publicly available for many species of Aspergillus.For example, GenBank® Accession Nos: AB326105.1 and BAG49047.1 discloseAspergillus terreus cadA nucleic acid and protein sequences,respectively (SEQ ID NOS: 49 and 50); GenBank® Accession Nos:XM_025706777.1 and XP_025563141.1 disclose Aspergillus vadensis CBS113365 cadA nucleic acid and protein sequences, respectively (SEQ IDNOS: 51 and 52); and GenBank® Accession Nos: XM_025663103.1 andXP_025520527.1 disclose Aspergillus piperis CBS 112811 cadA nucleic acidand protein sequences, respectively. However, one skilled in the artwill appreciate that in some examples, a cadA sequence can includevariant sequences (such as allelic variants and homologs) that retaincadA activity but when genetically inactivated in Aspergillus results ina fungus that has an ability to produce more aconitic acid than theparent strain without a genetically inactivated cadA gene (such as atleast 20%, at least 30%, at least 50%, at least 60%, at least 75%, atleast 100%, at least 200%, at least 500%, or 1000% more than a parentstrain under the same growing conditions, for example at day 5 ofproduction).

Detectable: Capable of having an existence or presence ascertained. Forexample, production of aconitic acid or 3-HP is detectable if the signalgenerated is strong enough to be measurable.

Exogenous: The term “exogenous” as used herein with reference to nucleicacid and a particular cell refers to any nucleic acid that does notoriginate from that particular cell as found in nature. Thus, anon-naturally-occurring nucleic acid is considered to be exogenous to acell once introduced into the cell. A nucleic acid that isnaturally-occurring also can be exogenous to a particular cell. Forexample, an entire chromosome isolated from cell X is an exogenousnucleic acid with respect to cell Y once that chromosome is introducedinto cell Y, even if X and Y are the same cell type.

In some examples, the panD, BAPAT, and HPDH nucleic acid or proteinexpressed in an Aspergillus terreus or Aspergillus pseudoterreus fungidoes not naturally occur in the Aspergillus terreus or Aspergilluspseudoterreus fungi and is therefore exogenous to that fungi. Forexample, the panD, BAPAT, and HPDH nucleic acid molecule introduced intoan Aspergillus terreus or Aspergillus pseudoterreus fungi can be fromanother organism, such as a bacterial panD, BAPAT, and HPDH sequence.

Genetic enhancement or up-regulation: When used in reference to theexpression of a nucleic acid molecule, such as a gene, refers to anyprocess which results in an increase in production of a gene product. Agene product can be RNA (such as mRNA, rRNA, tRNA, and structural RNA)or protein. Examples of processes that increase transcription includethose that facilitate formation of a transcription initiation complex,those that increase transcription initiation rate, those that increasetranscription elongation rate, those that increase processivity oftranscription and those that relieve transcriptional repression (forexample by blocking the binding of a transcriptional repressor). Geneup-regulation can include inhibition of repression as well as expressionabove an existing level. Examples of processes that increase translationinclude those that increase translational initiation, those thatincrease translational elongation and those that increase mRNAstability. In one example, additional copies of genes are introducedinto a cell in order to increase expression of that gene in theresulting transgenic cell.

Gene up-regulation includes any detectable increase in the production ofa gene product. In certain examples, production of a gene productincreases by at least 1.5-fold, at least 2-fold, or at least 5-fold),such as aspartate decarboxylase (panD), β-alanine-pyruvateaminotransferase (BAPAT), and 3-hydroxypropironate dehydrogenase (HPDH).For example, expression of a panD, BAPAT, and HPDH genes in Aspergillus(e.g., A. terreus) results in an Aspergillus strain having increasedlevels of the panD, BAPAT, and HPDH proteins, respectively, relative tothe parent strain, which can permit the recombinant fungus to produce3-HP. Genetic enhancement is also referred to herein as “enhancing orincreasing expression.”

Genetic inactivation or down-regulation: When used in reference to theexpression of a nucleic acid molecule, such as a gene, refers to anyprocess which results in a decrease in production of a gene product. Agene product can be RNA (such as mRNA, rRNA, tRNA, and structural RNA)or protein. Therefore, gene down-regulation or deactivation includesprocesses that decrease transcription of a gene or translation of mRNA.

For example, a mutation, such as a substitution, partial or completedeletion, insertion, or other variation, can be made to a gene sequencethat significantly reduces (and in some cases eliminates) production ofthe gene product or renders the gene product substantially or completelynon-functional. For example, a genetic inactivation of the cadA gene inAspergillus (e.g., A. pseudoterreus) results in Aspergillus having anon-functional or non-existent cadA protein, which results in therecombinant fungus to produce more aconitic acid. Genetic inactivationis also referred to herein as “functional deletion”.

Isolated: To be significantly separated from other agents. An “isolated”biological component (such as a nucleic acid molecule or protein) hasbeen substantially separated, produced apart from, or purified away fromother biological components in the cell of the organism in which thecomponent occurs, for example, other chromosomal and extra-chromosomalDNA and RNA, and proteins. Nucleic acid molecules and proteins whichhave been “isolated” include nucleic acid molecules and proteinspurified by standard purification methods. The term also embracesnucleic acid molecules and proteins prepared by recombinant expressionin a host cell as well as chemically synthesized proteins and nucleicacids. Samples of isolated biological components include samples of thebiological component wherein the biological component represents greaterthan 90% (for example, greater than 95%, such as greater than 98%) ofthe sample.

An “isolated” microorganism (such as a ΔcadA strain of Aspergillus) hasbeen substantially separated or purified away from microorganisms ofdifferent types, strains, or species. Microorganisms can be isolated bya variety of techniques, including serial dilution and culturing andresistance to certain chemicals, such as antibiotics. In some examples,an isolated AcadA strain of Aspergillus is at least 90% (for example, atleast 95%, as at least 98%, at least 99%, or at least 99.99%) pure.

Mutation: A change in a nucleic acid sequence (such as a gene sequence)or amino acid sequence, for example as compared to a nucleic acid oramino acid sequence present in a wild-type or native organism. Inparticular examples, a mutation is introduced into a cadA gene inAspergillus. Mutations can occur spontaneously, or can be introduced,for example using molecular biology methods (e.g., thereby generating arecombinant or transformed cell or microorganism). In particularexamples, a mutation includes one or more nucleotide substitutions,deletions, insertions, or combinations thereof. In particular examples,the presence of one or more mutations in a gene can significantlyinactivate and reduce expression of that gene.

Promoter: An array of nucleic acid control sequences which directtranscription of a nucleic acid. A promoter includes necessary nucleicacid sequences near the start site of transcription, such as, in thecase of a polymerase II type promoter, a TATA element. A promoter alsooptionally includes distal enhancer or repressor elements which can belocated as much as several thousand base pairs from the start site oftranscription. In some examples, a promoter is bi-directional. Nativeand non-native promoters can be used to drive expression of a gene, suchas panD, BAPAT, and HPDH. Exemplary promoters that can be used includebut are not limited to: eno1 promoter from A. niger, and dthl from A.nidulans or A. niger.

Examples of promoters include, but are not limited to the SV40 promoter,the CMV enhancer-promoter, and the CMV enhancer/β-actin promoter. Bothconstitutive and inducible promoters can be used in the methods providedherein (see e.g., Bitter et al., Methods in Enzymology 153:516-544,1987). Also included are those promoter elements which are sufficient torender promoter-dependent gene expression controllable for cell-typespecific, tissue-specific, or inducible by external signals or agents;such elements may be located in the 5′ or 3′ regions of the gene.Promoters produced by recombinant DNA or synthetic techniques can alsobe used to provide for transcription of the nucleic acid sequences.

Recombinant: A recombinant nucleic acid molecule or protein is one thathas a sequence that is not naturally occurring or has a sequence that ismade by an artificial combination of two otherwise separated segments ofsequence. In particular examples, this artificial combination isaccomplished by chemical synthesis or by the artificial manipulation ofisolated segments of nucleic acids, for example, by genetic engineeringtechniques such as those described in Sambrook et al. (ed.), MolecularCloning: A Laboratory Manual, 3d ed., vol. 1-3, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 2001. The term recombinantincludes nucleic acid molecules that have been altered solely byaddition, substitution, or deletion of a portion of the nucleic acidmolecule. A recombinant or transformed organism or cell, such as arecombinant Aspergillus, is one that includes at least one exogenousnucleic acid molecule, such as one used to genetically inactivate anendogenous cadA gene, and one used to express a non-native protein, suchas exogenous panD, BAPAT, and HPDH nucleic acid coding sequences.

Sequence identity/similarity: The identity/similarity between two ormore nucleic acid sequences, or two or more amino acid sequences, isexpressed in terms of the identity or similarity between the sequences.Sequence identity can be measured in terms of percentage identity; thehigher the percentage, the more identical the sequences are. Sequencesimilarity can be measured in terms of percentage similarity (whichtakes into account conservative amino acid substitutions); the higherthe percentage, the more similar the sequences are.

Methods of alignment of sequences for comparison are well known in theart. Various programs and alignment algorithms are described in: Smith &Waterman, Adv. Appl. Math. 2:482, 1981; Needleman & Wunsch, J. Mol.Biol. 48:443, 1970; Pearson & Lipman, Proc. Natl. Acad. Sci. USA85:2444, 1988; Higgins & Sharp, Gene, 73:237-44, 1988; Higgins & Sharp,CABIOS 5:151-3, 1989; Corpet et al., Nuc. Acids Res. 16:10881-90, 1988;Huang et al. Computer Appls. in the Biosciences 8, 155-65, 1992; andPearson et al., Meth. Mol. Bio. 24:307-31, 1994. Altschul et al., J.Mol. Biol. 215:403-10, 1990, presents a detailed consideration ofsequence alignment methods and homology calculations.

The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J.Mol. Biol. 215:403-10, 1990) is available from several sources,including the National Center for Biological Information (NCBI, NationalLibrary of Medicine, Building 38A, Room 8N805, Bethesda, Md. 20894) andon the Internet, for use in connection with the sequence analysisprograms blastp, blastn, blastx, tblastn and tblastx. Additionalinformation can be found at the NCBI web site.

BLASTN is used to compare nucleic acid sequences, while BLASTP is usedto compare amino acid sequences. To compare two nucleic acid sequences,the options can be set as follows: -i is set to a file containing thefirst nucleic acid sequence to be compared (e.g., C:\seq1.txt); -j isset to a file containing the second nucleic acid sequence to be compared(e.g., C:\seq2.txt); -p is set to blastn; -o is set to any desired filename (e.g., C:\output.txt); -q is set to −1; -r is set to 2; and allother options are left at their default setting. For example, thefollowing command can be used to generate an output file containing acomparison between two sequences: C:\B12seq -i c:\seq1.txt -jc:\seq2.txt -p blastn -o c:\output.txt -q −1 -r 2.

To compare two amino acid sequences, the options of B12seq can be set asfollows: -i is set to a file containing the first amino acid sequence tobe compared (e.g., C:\seq1.txt); -j is set to a file containing thesecond amino acid sequence to be compared (e.g., C:\seq2.txt); -p is setto blastp; -o is set to any desired file name (e.g., C:\output.txt); andall other options are left at their default setting. For example, thefollowing command can be used to generate an output file containing acomparison between two amino acid sequences: C:\B12seq -i c:\seq1.txt -jc:\seq2.txt -p blastp -o c:\output.txt. If the two compared sequencesshare homology, then the designated output file will present thoseregions of homology as aligned sequences. If the two compared sequencesdo not share homology, then the designated output file will not presentaligned sequences.

Once aligned, the number of matches is determined by counting the numberof positions where an identical nucleotide or amino acid residue ispresented in both sequences. The percent sequence identity is determinedby dividing the number of matches either by the length of the sequenceset forth in the identified sequence, or by an articulated length (e.g.,100 consecutive nucleotides or amino acid residues from a sequence setforth in an identified sequence), followed by multiplying the resultingvalue by 100. For example, a nucleic acid sequence that has 1166 matcheswhen aligned with a test sequence having 1554 nucleotides is 75.0percent identical to the test sequence (i.e., 1166÷1554*100=75.0). Thepercent sequence identity value is rounded to the nearest tenth. Forexample, 75.11, 75.12, 75.13, and 75.14 are rounded down to 75.1, while75.15, 75.16, 75.17, 75.18, and 75.19 are rounded up to 75.2. The lengthvalue will always be an integer. In another example, a target sequencecontaining a 20-nucleotide region that aligns with 20 consecutivenucleotides from an identified sequence as follows contains a regionthat shares 75 percent sequence identity to that identified sequence(i.e., 15÷20*100=75).

For comparisons of amino acid sequences of greater than about 30 aminoacids, the Blast 2 sequences function is employed using the defaultBLOSUM62 matrix set to default parameters, (gap existence cost of 11,and a per residue gap cost of 1). Homologs are typically characterizedby possession of at least 70% sequence identity counted over thefull-length alignment with an amino acid sequence using the NCBI BasicBlast 2.0, gapped blastp with databases such as the nr or swissprotdatabase. Queries searched with the blastn program are filtered withDUST (Hancock and Armstrong, 1994, Comput. Appl. Biosci. 10:67-70).Other programs use SEG. In addition, a manual alignment can beperformed. Proteins with even greater similarity will show increasingpercentage identities when assessed by this method, such as at least75%, 80%, 85%, 90%, 95%, or 99% sequence identity.

Nucleic acid sequences that do not show a high degree of identity maynevertheless encode identical or similar (conserved) amino acidsequences, due to the degeneracy of the genetic code. Changes in anucleic acid sequence can be made using this degeneracy to producemultiple nucleic acid molecules that all encode substantially the sameprotein. Such homologous nucleic acid sequences can, for example,possess at least 60%, 70%, 80%, 90%, 95%, 98%, or 99% sequence identitydetermined by this method.

One of skill in the art will appreciate that these sequence identityranges are provided for guidance only; it is possible that stronglysignificant homologs could be obtained that fall outside the rangesprovided. Thus, a variant cadA, panD, BAPAT, or HPDH protein or nucleicacid molecule that can be used with the organisims and methods of thepresent disclosure can have at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98% or at least 99% sequence identityto the SEQ ID NOs: and GenBank® Accession Nos. provided herein.

Transformed: A cell, such as a fungal cell, into which a nucleic acidmolecule has been introduced, for example by molecular biology methods.As used herein, the term transformation encompasses all techniques bywhich a nucleic acid molecule might be introduced into such a cell,including, but not limited to chemical methods (e.g., calcium-phosphatetransfection), physical methods (e.g., electroporation, microinjection,particle bombardment), fusion (e.g., liposomes), receptor-mediatedendocytosis (e.g., DNA-protein complexes, viral envelope/capsid-DNAcomplexes) and by biological infection by viruses such as recombinantviruses. In one example, the protoplast transformation provide herein,such as in Example 1, is used.

Vector: A nucleic acid molecule as introduced into a host cell, therebyproducing a transformed or recombinant host cell. A vector may includenucleic acid sequences that permit it to replicate in the host cell,such as an origin of replication. A vector may also include a panD,BAPAT, or HPDH coding sequence, or a sequence used to geneticallyinactivate cadA for example in combination with a promoter, and/orselectable marker genes, and other genetic elements. A vector cantransduce, transform or infect a cell, thereby causing the cell toexpress nucleic acids and/or proteins other than those native to thecell. A vector optionally includes materials to aid in achieving entryof the nucleic acid into the cell, such as a viral particle, liposome,protein coating or the like. In one example, a vector is a plasmid.

Overview

The filamentous fungus Aspergillus pseudoterreus has been used forindustrial production of itaconic acid. cis-aconitic acid decarboxylase(cadA) is the key enzyme in itaconic acid production. The itaconic acidbiosynthesis cluster is composed of genes tf, mttA, cadA and mfsA. Asshown in FIG. 1, itaconic acid (IA) is produced from glucose. Glucose isutilized in the cell mainly by the glycolytic pathway and metabolized topyruvate, which forms citric acid. cis-aconitic acid is derived fromcitric acid as a primary precursor of IA. cis-aconitic aciddecarboxylase (cadA) removes carbon dioxide from cis-aconitic acid andforms itaconic acid. However, cadA is localized in the cytosol, whilecis-aconitic acid is formed from the TCA cycle in the mitochondria. mttAis localized on the mitochondrial membrane and functiond to transportcis-aconitic acid from mitochondria to cytosol. Another transporter,mfsA is also an organic acid transporter that may be involved inexporting itaconic acid out of cells.

The first demonstration of genetically inactivating the cadA gene inAspergillus pseudoterreus is shown herein. In the cadA deletion strain(ΔcadA), no more itaconic acid is produced. At the same time significantamount of cis-aconitic acid and trans-aconitic acid are detected.Blocking the itaconic acid production pathway permits the carbon to bediverted towards other organic acid production. The ΔcadA Aspergilluscan be used as a host for chemical platform, and provides a new way toproduce aconitic acid and other organic acids (for example by expressingother genes needed for procution of those acids, such as panD, BAPAT,and HPDH for 3-HP production). This strain works as biocatalyst thatconverts biomass into aconitic acid through bioproduction method at roomtemperature (such as about 20-35° C.) and ordinary pressure (such asabout 1 atm). Current processes of aconitic acid production includechemical synthesis that require high temperatures and harmful reagents.

The EST data provided herein demonstrated that four genes, tf, cadA,mttA and mfsA show high transcription frequency after IA productionstarts, but not before IA production begins. The high expression ofthese genes persists through the production process. Genes upstream anddownstream of the cluster did not show expression differences before andafter production. One gene downstream next to mfsA, a p450 enzyme, alsoshowed high expression after IA production started, however, deletion ofthis gene did not effect IA yield.

Correlations between the IA gene cluster and IA production were furtherinvestigated by constructing deletion strains. In a Δcad strain, no IAwas detected, while trace amounts of IA were detected in an mttAknockout. IA production in an mfsA deletion strain decreased one thirdcompared with wild type. This indicates mfsA can transport IA across thecell membrane. In the Δtf strain, IA production decreased eight fold andslowed the production rate compared to wild type. Also in the tfdeletion strain, expression of cadA, mttA and mfsA significantlydecreased. RT-PCR results indicated that the expression level of genesin the IA cluster was regulated by tf, which is turned on by IAproduction conditions.

The ΔcadA strain produced aconitc acid. During the production,cis-aconitic acid was detected first, followed by the appearance oftrans-aconitic acid. cis-aconitic acid levels remained consistent fromday 5 forward. The trans-aconitic acid levels continued to increase fromdays 4 to 10. By day 10, more than 10g/L trans-aconitic acid wasdetected in the supernatant. In the ΔcadA strain, cis-aconitic aciddecarboxylase is not produced, and the cis-aconitic acid cannot beconverted to itaconic acid by decarboxylation and accumulates in thecell. cis-aconitic acid was transported outside the cell. cis-aconiticacid is not stable in the acid solution and is rapidly converted intotrans-aconitic acid.

Aconitic acid is an unsaturated tricarcoxylic acid and is noted as a top30 potential building block by United States Department of Energy (DOE).Trans-aconitic acid can be used to make polymers. Currently,trans-aconitc acid is produced by chemical synthesis and requires hightemperature and harmful solvents. Generation of trans-aconitic acid hasbeen achieved by metabolic engineering aconitase isomerase fromPseudomonas sp. WU-0701 into E. coli. However, the substrate for therecombinant E. coli to produce trans-aconitic acid is citric acid, whichhas to be generated first from fermentation. In contrast, the disclosedΔcadA fungi can produce trans-aconitic acid directly from renewablebiomass substrates. Also since the cadA is not functional and precursorsfrom TAC cycle accumulate in the cell, the carbon can be rerouted togenerate other organic acid since A. pseudoterreus is industrialfilamentous fungi and tolerant to low pH.

Based on these observations, provided herein are isolated recombinant(i.e., transformed) Aspergillus fungi that include a geneticinactivation (also referred to as a functional deletion) of anendogenous cis-aconitic acid decarboxylase (cadA) gene. Such fungi arereferred to herein as ΔcadA fungi. Exemplary Aspergillus species thatcan be used include Aspergillus pseudoterreus and Aspergillus terreus.In some examples, the endogenous cadA gene is genetically inactivated bymutation (such as a complete or partial deletion of the cadA gene) or byinsertional mutation (such as by insertion of another nucleic acidmolecule into the cadA gene, such as an antibiotic resistance marker).

In some examples, the cadA gene prior to its genetic inactivationencodes a protein having at least 80%, at least 90%, at least 95%, atleast 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 50 or52. In some examples, the cadA gene (or its coding sequence) prior toits genetic inactivation comprises at least 80%, at least 90%, at least95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:49, 51, 59 or 92.

The disclosed ΔcadA fungi can include other exogenous genes to expressproteins needed to permit the fungi to produce other organic acids. Forexample, the disclosed ΔcadA fungi can further include an exogenousnucleic acid molecule encoding aspartate 1-decarboxylase (panD), anexogenous nucleic acid molecule encoding β-alanine-pyruvateaminotransferase (BAPAT), and an exogenous nucleic acid moleculeencoding 3-hydroxypropionate dehydrogenase (HPDH). panD, BAPAT, and HPDHcoding sequences can be part of a one or more nucleic acid molecules,such as a vector. In addition, expression of the panD, BAPAT, and HPDHcoding sequences can be driven by one or more promoters, such as abi-directional promoter. In some examples, the promoter is native to thegene it is expressing. In some examples, the promoter is from A. niger.In some examples, the panD, BAPAT, and/or HPDH coding sequences areinserted into the cadA gene, genetically inactivating cadA. In someexamples, the exogenous nucleic acid molecule encoding panD has at least80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 53 or 65, and/or encodes a panD proteincomprising at least 80%, at least 90%, at least 95%, at least 98%, atleast 99%, or 100% sequence identity to SEQ ID NO: 54. In some examples,the exogenous nucleic acid molecule encoding BAPAT has at least 80%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100% sequenceidentity to SEQ ID NO: 55, and/or encodes a BAPAT protein having atleast 80%, at least 90%, at least 95%, at least 98%, at least 99%, or100% sequence identity to SEQ ID NO: 56. In some examples, the exogenousnucleic acid molecule encoding HPDH has at least 80%, at least 90%, atleast 95%, at least 98%, at least 99%, or 100% sequence identity to SEQID NO: 57, and/or encodes a HPDH protein having at least 80%, at least90%, at least 95%, at least 98%, at least 99%, or 100% sequence identityto SEQ ID NO: 58.

The disclosure also provides compositions that include the ΔcadA fungi,and the ΔcadA fungi expressing other genes (such as panD, BAPAT, andHPDH). Such a composition can include a solid or liquid culture orgrowth media, such as complete media, minimal media, or Riscaldatimedium (such as modified Riscaldati medium with 20× trace elements).

The disclosure also provides kits that include the ΔcadA fungi, and theΔcadA fungi expressing other genes (such as panD, BAPAT, and HPDH). Sucha kits can include a solid or liquid culture or growth media, such ascomplete media, minimal media, or Riscaldati medium (such as modifiedRiscaldati medium with 20× trace elements).

Also provided are methods of using the disclosed ΔcadA fungi to makeaconitic acid. Such a method can include culturing the recombinantAspergillus ΔcadA fungi under conditions that permit the fungus to makeaconitic acid, such as growth in Riscaldati medium, thereby makingaconitic acid. In some examples the aconitic acid generated iscis-aconitic acid, trans-aconitic acid, or both. In some examples, thefungi are cultured at room temperature (e.g., 20-35° C.) at normalatmospheric pressure (e.g., 1 atm). In some examples, the methodincludes purifying or isolating the aconitic acid, for example from theculture media or from the cultured fungus. In some examples, theaconitic acid is isolated at least 2 days, at least 3 days, at least 5days, at least 8 days or at least 10 days after the start of culturing,such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19or 20 days after the start of culturing.

Also provided are methods of using the disclosed ΔcadA fungi expressingpanD, BAPAT, and HPDH to make 3-HP. Such a method can include culturingthe recombinant Aspergillus ΔcadA fungi expressing panD, BAPAT, and HPDHunder conditions that permit the fungus to make 3-HP, such as growth inRiscaldati medium (such as modified Riscaldati medium with 20× traceelements), thereby making 3-HP. In some examples, the fungi are culturedat room temperature (e.g., 20-35° C.) at normal atmospheric pressure(e.g., 1 atm). In some examples, the method includes purifying orisolating the 3-HP, for example from the culture media or from thecultured fungus. In some examples, the 3-HP is isolated at least 2 days,at least 3 days, at least 5 days, at least 8 days or at least 10 daysafter the start of culturing, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19 or 20 days after the start of culturing.

Recombinant ΔcadA Fungi

The present disclosure provides isolated recombinant Aspergillus fungihaving its endogenous cadA gene genetically inactivated (e.g.,functional deletion) of. Such fungi are referred to herein as ΔcadAfungal strains. It is shown herein that ΔcadA Aspergillus strains haveincreased aconitic acid production as compared to Aspergillus havingnative levels of cadA expression.

Any variety or strain of Aspergillus can be used. In particularexamples, the Aspergillus fungus is A. terreus or A. pseudoterreus, aswell as particular strains thereof (for example A. terreus NRRL 1960, A.pseudoterreus ATCC 32359).

In addition, any method for genetic inactivation can be used, as long asthe expression of the cadA gene is significantly reduced or eliminated,or the function of the cadA protein is significantly reduced oreliminated. In particular examples, the cadA gene is geneticallyinactivated by complete or partial deletion mutation or by insertionalmutation. In some examples genetic inactivation need not be 100%. Insome embodiments, genetic inactivation refers to at least 50%, at least60%, at least 70%, at least 80%, at least 90%, or at least 95% gene orprotein inactivation. The term “reduced” or “decreased” as used hereinwith respect to a cell and a particular gene or protein activity refersto a lower level of activity than that measured in a comparable cell ofthe same species. For example, a particular A. terreus or A.pseudoterreus lacking cadA activity has reduced cadA activity if acomparable A. terreus or A. pseudoterreus not having an cadA geneticinactivation has detectable cadA activity.

cadA sequences are disclosed herein and others are publicly available,for example from GenBank or EMBL. In some examples, the cadA genefunctionally deleted encoded a protein having at least 80%, at least90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 50 or 52 prior to is geneticinactivation. In some examples, the endogenous cadA gene functionallydeleted comprises at least 80%, at least 90%, at least 95%, at least97%, or at least 98% sequence identity to SEQ ID NO: 49, 51, 59, or 92prior to is genetic inactivation.

The genetic inactivation of cadA results in many phenotypes in therecombinant ΔcadA Aspergillus, such as A. terreus or A. pseudoterreus.For example, ΔcadA mutants can have one or more of the followingphenotypes: produces at least 2-fold, at least 3-fold,at least 3.5 fold,at least 5-fold, at least 8-fold, or at least 10-fold more totalaconitic acid than a wild-type Aspergillus terreus or Aspergilluspseudoterreus (for example at day 3, 4, 5, 6, 7, 8, 9 or 10 ofproduction); produces at least 2-fold more cis-aconitic acid at day 5,6, 7, 8, 9, or 10 of culturing in Riscaldati medium than a wild-typeAspergillus terreus or Aspergillus pseudoterreus; produces at least2-fold, at least 3-fold, at least 5-fold, or at least 10-fold moretrans-aconitic acid at day 10 of culturing in Riscaldati medium than awild-type Aspergillus terreus or Aspergillus pseudoterreus; orcombinations thereof. In some examples, such increases are relative toAspergillus terreus strain ATCC 32359 grown under the same conditions asthe ΔcadA mutant. In some examples, an increased total aconitic acidproduction by ΔcadA fungi occurs at least 3 days (such as at least 4, 5,6, 7, 8, 9, or 10 days) after inoculation in Riscaldati medium (such asat least 0.5 g/L aconitic acid or at least 1 g/L aconitic acid), ascompared to no detectable aconitic acid produced by Aspergillus terreusstrain ATCC 32359 at the same time point.

Additional genes can also be inactivated in the ΔcadA fungi, wherein theadditional genes may or may not provide additional enhancement ofaconitic acid production to the fungus. In one example, the ΔcadA fungiincludes overexpressed or upregulated aconitic acid transporters.

In some examples, ΔcadA fungi include one or more additional exogenousnucleic acid molecules, for example to permit production of otherorganic acids by the recombinant fungi. In one example, the ΔcadA fungiincludes an exogenous nucleic acid molecule encoding aspartatedecarboxylase (panD), an exogenous nucleic acid molecule encodingβ-alanine-pyruvate aminotransferase (BAPAT), and an exogenous nucleicacid molecule encoding 3-hydroxypropironate dehydrogenase (HPDH). Suchexogenous nucleic acid molecules can be part of one or more exogenousnucleic acid molecules (such as 1, 2 or 3 exogenous nucleic acidmolecules). In some examples, exogenous nucleic acid molecules can bepart of a vector, such as a plasmid or viral vector. In some examples,expression of the exogenous nucleic acid molecules is driven by one ormore promoters, such as a constitutive or inducible promoter, or abi-directional promoter. In some examples, the promoter used to driveexpression of panD, BAPAT, and HPDH is a native promoter (e.g., nativeto the panD, BAPAT, and HPDH gene expressed). In other examples, thepromoter used to drive expression of panD, BAPAT, and HPDH is anon-native promoter (e.g., exogenous to the panD, BAPAT, and HPDH geneexpressed). In some examples, such a ΔcadA fungi expressing panD, BAPAT,and HPDH are used to produce 3-HP.

A. Methods of Functionally Deleting cadA

As used herein, an “inactivated” or “functionally deleted” cadA genemeans that the cadA gene has been mutated, for example by insertion,deletion, or substitution (or combinations thereof) of one or morenucleotides such that the mutation substantially reduces (and in somecases abolishes) expression or biological activity of the encoded cadAgene product. The mutation can act through affecting transcription ortranslation of the cadA gene or its mRNA, or the mutation can affect thecadA polypeptide product itself in such a way as to render itsubstantially inactive.

In one example, a strain of Aspergillus is transformed with a vectorwhich has the effect of down-regulating or otherwise inactivating a cadAgene. This can be done by mutating control elements such as promotersand the like which control gene expression, by mutating the codingregion of the gene so that any protein expressed is substantiallyinactive, or by deleting the cadA gene entirely. For example, a cadAgene can be functionally deleted by complete or partial deletionmutation (for example by deleting a portion of the coding region of thegene) or by insertional mutation (for example by inserting a sequence ofnucleotides into the coding region of the gene, such as a sequence ofabout 1-5000 nucleotides). In one example, the cadA gene is geneticallyinactivated by inserting coding sequences for panD, BAPAT, and/or HPDH.Thus, the disclosure provides transformed fungi that include at leastone exogenous nucleic acid molecule which genetically inactivates a cadAgene. In one example, such a transformed cell produces more aconiticacid, for example relative to a comparable fungus with a native orwild-type cadA sequence.

In particular examples, an insertional mutation includes introduction ofa sequence that is in multiples of three bases (e.g., a sequence of 3,9, 12, or 15 nucleotides) to reduce the possibility that the insertionwill be polar on downstream genes. For example, insertion or deletion ofeven a single nucleotide that causes a frame shift in the open readingframe, which in turn can cause premature termination of the encoded cadApolypeptide or expression of a substantially inactive polypeptide.Mutations can also be generated through insertion of foreign genesequences, for example the insertion of a gene encoding antibioticresistance (such as hygromycin or bleomycin), or panD, BAPAT, and/orHPDH coding sequences.

In one example, genetic inactivation is achieved by deletion of aportion of the coding region of the cadA gene. For example, some, most(such as at least 50%) or virtually the entire coding region can bedeleted. In particular examples, about 5% to about 100% of the gene isdeleted, such as at least 20% of the gene, at least 40% of the gene, atleast 75% of the gene, or at least 90% of the cadA gene.

Deletion mutants can be constructed using any of a number of techniques.In one example, homologous double crossover with fusion PCR products isemployed to genetically inactivate one or more genes in Aspergillus. Aspecific example of such a method is described in Example 1 below.

In one example, a strategy using counterselectable markers can beemployed which has been utilized to delete genes. For a review, seeReyrat et al. (Infec. Immun. 66:4011-4017, 1998). In this technique, adouble selection strategy is employed wherein a plasmid is constructedencoding both a selectable and counterselectable marker, with flankingDNA sequences derived from both sides of the desired deletion. Theselectable marker is used to select for fungi in which the plasmid hasintegrated into the genome in the appropriate location and manner. Thecounterselecteable marker is used to select for the very smallpercentage of fungi that have spontaneously eliminated the integratedplasmid. A fraction of these fungi will then contain only the desireddeletion with no other foreign DNA present.

In another technique, the cre-lox system is used for site specificrecombination of DNA (for example see Steiger et al., Appl. Environ.Microbiol. 77(1):114, 2011). The system includes 34 base pair loxsequences that are recognized by the bacterial cre recombinase gene. Ifthe lox sites are present in the DNA in an appropriate orientation, DNAflanked by the lox sites will be excised by the cre recombinase,resulting in the deletion of all sequences except for one remaining copyof the lox sequence. Using standard recombination techniques, thetargeted gene of interest (e.g., cadA) can be deleted in the Aspergillusgenome and to replace it with a selectable marker (for example a genecoding for kanamycin resistance) that is flanked by the lox sites.Transient expression (by electroporation of a suicide plasmid containingthe cre gene under control of a promoter that functions in Aspergillus)of the cre recombinase should result in efficient elimination of the loxflanked marker. This process will produce a mutant containing thedesired deletion mutation and one copy of the lox sequence.

In another method, a cadA gene sequence in the Aspergillus genome isreplaced with a marker gene, such as green fluorescent protein,β-galactosidase, or luciferase. In this technique, DNA segments flankinga desired deletion are prepared by PCR and cloned into a suicide(non-replicating) vector for Aspergillus. An expression cassette,containing a promoter active in Aspergillus and the appropriate markergene, is cloned between the flanking sequences. The plasmid isintroduced into wild-type Aspergillus. Fungi that incorporate andexpress the marker gene are isolated and examined for the appropriaterecombination event (replacement of the wild type cadA gene with themarker gene).

Thus, for example, a fungal cell can be engineered to have a disruptedcadA gene using common mutagenesis or knock-out technology. (Methods inYeast Genetics (1997 edition), Adams, Gottschling, Kaiser, and Sterns,Cold Spring Harbor Press, 1998; Datsenko and Wanner, Proc. Natl. Acad.Sci. USA 97: 6640-5, 2000; and Dai et al., Appl. Environ. Microbiol.70(4):2474-85, 2004). Alternatively, antisense technology can be used toreduce or eliminate the activity of cadA. For example, a fungal cell canbe engineered to contain a cDNA that encodes an antisense molecule thatprevents cadA from being translated. The term “antisense molecule”encompasses any nucleic acid molecule or nucleic acid analog (e.g.,peptide nucleic acids) that contains a sequence that corresponds to thecoding strand of an endogenous cadA gene. An antisense molecule also canhave flanking sequences (e.g., regulatory sequences).

Thus, antisense molecules can be ribozymes or antisenseoligonucleotides. A ribozyme can have any general structure including,without limitation, hairpin, hammerhead, or axehead structures, providedthe molecule cleaves RNA. Further, gene silencing can be used to reducethe activity of cadA.

In one example, to genetically inactivate cadA in A. pseudoterreus or A.terreus, protoplast transformation is used, for example as described inExample 1. For example, conidia of A. pseudoterreus or A. terreus aregrown in liquid complete medium at room temperature (e.g., about 20-35°C., such as 30° C.) and grown for at least 12 hours (such as at least 16hours, or at least 18 hours, such as 12-24 hours, or 16-18 hours), atleast 100 rpm, such as at least 150 rpm, for example 100 to 200 rpm. Theresulting mycelia are subsequently harvested, for example by filtration.Protoplasts are prepared, for example by treating the harvested myceliawith a lysing enzyme (for example in an osmotic wash buffer for at least30 min, at least 60 min, at least 120 min, or at leave 240 min, such as2 h). The resulting protoplasts are collected (e.g., by filtering).Protoplasts can be washed, for example with a Washing Solution (0.6MKCl, 0.1M Tris/HCl, pH 7.0) and Conditioning Solution (0.6M KCl, 50 mMCaCl₂, 10 mM Tris/HCl, pH 7.5). The protoplasts are transformed, forexample in the conditioning solution. In some examples, at least 0.5 ug,at least 1 ug, or at least 2 ug of DNA (such as 1-2 ug DNA) is added toat least 10⁶ protoplasts (such as at least 10⁷ or 2×10⁷ protoplasts).Polyethylene glycol (PEG), such as PEG8000 is added (such as 25%PEG8000, 0.6M KCl, 50 mM CaCl₂, 10 mM Tris/HCl, and pH 7.5) and thereaction incubated for at least 5 min (such as at least 10 min, at least20 min, or at least 30 min, such as 10-30 min, 15-20 min, or 20 min) onice. Additional PEG solution can be added and the reaction incubated forat least 1 min, at least 3 min, or at least 5 min, on ice. ConditioningSolution is added to the reaction, and the protoplast suspension mixedwith warm selection agar (Minimal media+0.6M KCl+1.5% Agar+100 ug/mlhygromycin) (such as at 50° C.), and poured directly onto petri dishplates and allowed to solidify. Solidified plates can be inverted andincubated overnight at room temperature (e.g., about 20-35° C., such as30° C.). The following day, the plates can be overlaid with MinimalMedium containing a selection antibiotic, such as hygromycin. Coloniesappear after 3-4 days. Transformants can be excised and transferred toMM plate containing the selection antibiotic.

B. Measuring Gene Inactivation

A fungus having an inactivated cadA gene can be identified using knownmethods. For example, PCR and nucleic acid hybridization techniques,such as Northern and Southern analysis, can be used to confirm that afungus has a genetically inactivated cadA gene. In one example,real-time reverse transcription PCR (qRT-PCR) is used for detection andquantification of targeted messenger RNA, such as mRNA of cadA gene inthe parent and mutant strains as grown at the same culture conditions.Immunohisto-chemical and biochemical techniques can also be used todetermine if a cell expresses cadA by detecting the expression of thecadA peptide encoded by cadA. For example, an antibody havingspecificity for cadA can be used to determine whether or not aparticular fungus contains a functional nucleic acid encoding cadAprotein. Further, biochemical techniques can be used to determine if acell contains a cadA gene inactivation by detecting a product producedas a result of the lack of expression of the peptide. For example,production of aconitic acid by A. terreus or A. pseudoterreus canindicate that such a fungus contains an inactivated cadA gene.

C. Measuring Aconitic Acid Production

Methods of determining whether a genetic inactivation of cadA inAspergillus, such as A. terreus or A. pseudoterreus. increases aconiticacid production, for example relative to the same strain of A. terreusor A. pseudoterreus with a native cadA sequence (such as a parentalstrain), are provided herein. Although particular examples are disclosedherein, the methods are not limiting.

For example, production of aconitic acid by Aspergillus (such as a ΔcadAstrain) can be measured using a spectrophotometric assay, by liquidchromatography (LC), or high-pressure liquid chromatography (HPLC)methods. In some examples, the supernatant of the fungus is analyzed forthe presence of aconitic acid. In some examples, the culture mediacontaining the ΔcadA strain is filtered prior to measuring aconitic acidin the culture media (supernatant).

D. cadA Sequences

cadA protein and nucleic acid sequences are publicly available andspecific examples are provided herein. In addition, cadA sequences canbe identified using molecular biology methods.

Examples of cadA nucleic acid sequences are shown in SEQ ID NOS: 49, 51,59 and 92. However, the disclosure also encompasses variants of SEQ IDNOS: 49, 51, 59 and 92 which encode a functional cadA protein. Oneskilled in the art will understand variants of the cadA nucleic acidsequences provided herein can be genetically inactivated. Variantsequences may contain a single insertion, a single deletion, a singlesubstitution, multiple insertions, multiple deletions, multiplesubstitutions, or any combination thereof (e.g., single deletiontogether with multiple insertions). In addition, the degeneracy of thecode permits multiple nucleic acid sequences to encode the same protein.Such variant cadA nucleic acid molecules can share at least 80%, atleast 85%, at least 90%, at least 95%, at least 97%, at least 98%, or atleast 99% sequence identity to any cadA nucleic acid sequence, such asSEQ ID NO: 49, 51, 59 or 92.

Examples of cadA protein sequences are shown in SEQ ID NOS: 50 and 52.However, the disclosure also encompasses variants SEQ ID NOS: 50 and 52which retain cadA activity. One skilled in the art will understand thatvariants of these cadA enzyme sequences can be inactivated. Variantsequences can be identified, for example by aligning known cadAsequences. Variant sequences may contain a single insertion, a singledeletion, a single substitution, multiple insertions, multipledeletions, multiple substitutions, or any combination thereof (e.g.,single deletion together with multiple insertions). Such cadA peptidesshare at least 80%, at least 85%, at least 90%, at least 95%, at least97%, at least 98%, or at least 99% sequence identity to a cadA proteinsequence, such as SEQ ID NO: 50 or 52.

In some examples, a cadA sequence that is to be genetically inactivatedencodes or includes one or more conservative amino acid substitutions. Aconservative amino acid substitution is a substitution of one amino acid(such as one found in a native sequence) for another amino acid havingsimilar biochemical properties. Typically, conservative substitutionshave little to no impact on the activity of a resulting peptide. In oneexample, a cadA sequence (such as SEQ ID NO: 50 or 52) includes one ormore amino acid substitutions, such as conservative substitutions (forexample at 1, 2, 5 or 10 residues). Examples of amino acids which may besubstituted for an original amino acid in a protein and which areregarded as conservative substitutions include: Ser for Ala; Lys forArg; Gln or His for Asn; Glu for Asp; Ser for Cys; Asn for Gln; Asp forGlu; Pro for Gly; Asn or Gln for His; Leu or Val for Ile; Ile or Val forLeu; Arg or Gln for Lys; Leu or Ile for Met; Met, Leu or Tyr for Phe;Thr for Ser; Ser for Thr; Tyr for Trp; Trp or Phe for Tyr; and Ile orLeu for Val. Further information about conservative substitutions can befound in, among other locations in, Ben-Bassat et al., (J. Bacteriol.169:751-7, 1987), O′Regan et al., (Gene 77:237-51, 1989), Sahin-Toth etal., (Protein Sci. 3:240-7, 1994), Hochuli et al., (Bio/Technology6:1321-5, 1988), WO 00/67796 (Curd et al.) and in standard textbooks ofgenetics and molecular biology.

The cadA gene inactivated in a fungus, in particular examples, includesa sequence that encodes a cadA protein having at least at least 80%, atleast 85%, at least 90%, at least 95%, at least 97%, at least 98%, atleast 99%, or 100% sequence identity to a cadA protein sequence, such asSEQ ID NO: 50 or 52, wherein the protein can catalyze thedecarboxylation of cis-aconitate to itaconate and CO₂ and vice versa. Ina specific example, the cadA gene inactivated in a fungus encodes a cadAprotein shown in SEQ ID NO: 50 or 52.

The cadA gene that is to be inactivated in a fungus, in particularexamples, includes a sequence (such as a coding sequence) having atleast at least 80%, at least 85%, at least 90%, at least 95%, at least97%, at least 98%, at least 99%, or 100% sequence identity to a cadAnucleic acid sequence, such as SEQ ID NO: 49, 51, 59, or 92, and encodesa cadA protein that can catalyze the decarboxylation of cis-aconitate toitaconate and CO₂ and vice versa. In a specific example, cadA geneinactivated in a fungus is the sequence of SEQ ID NO: 2 or 4.

One skilled in the art will appreciate that additional cadA sequencescan be identified. For example, cadA nucleic acid molecules that encodea cadA protein can be identified and obtained using molecular cloning orchemical nucleic acid synthesis procedures and techniques, includingPCR. In addition, nucleic acid sequencing techniques and softwareprograms that translate nucleic acid sequences into amino acid sequencesbased on the genetic code can be used to determine whether or not aparticular nucleic acid has any sequence homology with known cadAsequences. Sequence alignment software such as MEGALIGN (DNASTAR,Madison, Wis., 1997) can be used to compare various sequences.

In addition, nucleic acid hybridization techniques can be used toidentify and obtain a nucleic acid molecule that encodes a cadA protein.Briefly, any known cadA nucleic acid molecule, or fragment thereof, canbe used as a probe to identify similar nucleic acid molecules byhybridization under conditions of moderate to high stringency. Suchsimilar nucleic acid molecules then can be isolated, sequenced, andanalyzed to determine whether the encoded protein is a cadA protein.

E. panD, BAPAT, and HPDH Sequences

panD, BAPAT, and HPDH protein and nucleic acid sequences are publiclyavailable and specific examples are provided herein. In addition, panD,BAPAT, and HPDH sequences can be identified using molecular biologymethods.

Exemplary of panD coding sequences are shown in SEQ ID NO: 53 and 65.However, the disclosure also encompasses variants of SEQ ID NO: 53 and65 which encode a functional panD protein. Exemplary of BAPAT codingsequences are shown in SEQ ID NO: 55 and 71. However, the disclosurealso encompasses variants of SEQ ID NO: 55 and 71 which encode afunctional BAPAT protein. Exemplary of HPDH coding sequences are shownin SEQ ID NO: 57 and 80. However, the disclosure also encompassesvariants of SEQ ID NO: 57 and 80 which encode a functional HPDH protein.

One skilled in the art will understand variants of the panD, BAPAT, andHPDH nucleic acid sequences provided herein can be introduced into anAspergillus fungus, such as one that is ΔcadA, such as inserting panD,BAPAT, and HPDH expression sequences into the native cadA gene.toinactivate it. Variant sequences may contain a single insertion, asingle deletion, a single substitution, multiple insertions, multipledeletions, multiple substitutions, or any combination thereof (e.g.,single deletion together with multiple insertions). In addition, thedegeneracy of the code permits multiple nucleic acid sequences to encodethe same protein. In some examples, a panD, BAPAT, and HPDH sequencethat is to be expressed in an Aspergillus fungus is codon optimized forexpression in Aspergillus, such as Aspergillus terreus or pseudoterreus.Such variant panD, BAPAT, and HPDH nucleic acid molecules in someexamples share at least 80%, at least 85%, at least 90%, at least 95%,at least 97%, at least 98%, or at least 99% sequence identity to anypanD, BAPAT, and HPDH nucleic acid sequence, such as SEQ ID NO: 53, 55,or 57, respectively, or SEQ ID NO: 65, 71, or 80, respectively.

Exemplary panD, BAPAT, and HPDH protein sequences are shown in SEQ IDNOS: 54, 56, and 58, respectively. However, the disclosure alsoencompasses variants SEQ ID NOS: 54, 56, and 58 which retain panD,BAPAT, and HPDH activity, respectively. One skilled in the art willunderstand that variants of these panD, BAPAT, and HPDH sequences can beexpressed in an Aspergillus fungus, such as one that is ΔcadA, Variantsequences can be identified, for example by aligning known panD, BAPAT,and HPDH sequences. Variant sequences may contain a single insertion, asingle deletion, a single substitution, multiple insertions, multipledeletions, multiple substitutions, or any combination thereof (e.g.,single deletion together with multiple insertions). Such panD, BAPAT,and HPDH peptides expressed in aΔcadA fungus in some examples share atleast 80%, at least 85%, at least 90%, at least 95%, at least 97%, atleast 98%, or at least 99% sequence identity to a panD, BAPAT, and HPDHprotein sequence, such as SEQ ID NO: 54, 56, or 58, respectively.

In some examples, a panD, BAPAT, and HPDH sequence that is to beexpressed in an Aspergillus fungus encodes or includes one or moreconservative amino acid substitutions. In one example, a panD, BAPAT, orHPDH sequence (such as SEQ ID NO: 54, 56, or 58, respectively) includesone or more amino acid substitutions, such as conservative substitutions(for example at 1, 2, 5, or 10 residues). Examples of conservativesubstitutions are provided elsewhere herein.

The panD, BAPAT, and HPDH gene expressed in a fungus, in particularexamples, includes a sequence that encodes a panD, BAPAT, and HPDHprotein having at least at least 80%, at least 85%, at least 90%, atleast 95%, at least 97%, at least 98%, at least 99%, or 100% sequenceidentity to a panD, BAPAT, and HPDH protein sequence, such as SEQ ID NO:54, 56, or 58, respectively, wherein the variant protein has thebiological activity of panD, BAPAT, or HPDH , respectively. In aspecific example, the panD, BAPAT, and HPDH gene expressed in a ΔcadAfungus encodes the protein shown in SEQ ID NO: 54, 56, and 58,respectively.

One skilled in the art will appreciate that additional panD, BAPAT, andHPDH sequences can be identified. For example, panD, BAPAT, and HPDHnucleic acid molecules that encode a panD, BAPAT, and HPDH protein,respectively can be identified and obtained using molecular cloning orchemical nucleic acid synthesis procedures and techniques, includingPCR. In addition, nucleic acid sequencing techniques and softwareprograms that translate nucleic acid sequences into amino acid sequencesbased on the genetic code can be used to determine whether or not aparticular nucleic acid has any sequence homology with panD, BAPAT, orHPDH sequences. Sequence alignment software such as MEGALIGN (DNASTAR,Madison, Wis., 1997) can be used to compare various sequences.

In addition, nucleic acid hybridization techniques can be used toidentify and obtain a nucleic acid molecule that encodes a panD, BAPAT,or HPDH protein. Briefly, any known panD, BAPAT, or HPDH nucleic acidmolecule, or fragment thereof, can be used as a probe to identifysimilar nucleic acid molecules by hybridization under conditions ofmoderate to high stringency. Such similar nucleic acid molecules thencan be isolated, sequenced, and analyzed to determine whether theencoded protein is a panD, BAPAT, or HPDH protein.

In one example, exogenous panD, BAPAT, and/or HPDH nucleic acidsequences are introduced into A. pseudoterreus or A. terreus usingprotoplast transformation, for example as described in Example 1 (anddescribed above).

F. Methods of Increasing panD, BAPAT, and HPDH Expression

In some examples, a native A. pseudoterreus or A. terreus fungi does nothave or express panD, BAPAT, and/or HPDH nucleic acid sequences. Thus,in some examples, expression of these genes is increased by introducingpanD, BAPAT, and/or HPDH nucleic acid coding sequences (such may becodon optimized) into the A. pseudoterreus or A. terreus fungi.

In some examples, a native A. pseudoterreus or A. terreus fungi doesexpress native panD, BAPAT, and/or HPDH nucleic acid sequences. Thus, insome examples, expression of these genes is upregulated by introducingadditional copies of panD, BAPAT, and/or HPDH nucleic acid codingsequences (such may be codon optimized) into the A. pseudoterreus or A.terreus fungi. As used herein, “up-regulated” gene means that expressionof the gene or gene product (e.g., protein) has been up-regulated, forexample by introduction of additional copies of the appropriate gene orcoding sequence into the fungus (or other molecular biology methods),such that the introduced nucleic acid sequence is expressed, resultingin increased expression or biological activity of the encoded geneproduct. In some embodiments, introduction of one or more transgenesincluding panD, BAPAT, and/or HPDH coding sequences into a native A.pseudoterreus or A. terreus fungi increases expression of panD, BAPAT,and/or HPDH by at least 20%, at least 40%, at least 50%, at least 100%,at least 150%, at least 200%, at least 300%, or at least 500%, forexample relative to the parental fungal strain without the introducedpanD, BAPAT, and/or HPDH coding sequences. The term “increased” or“up-regulated” as used herein with respect to a cell and a particulargene or protein activity refers to a higher level of activity than thatmeasured in a comparable cell of the same species. For example, aparticular fungi having increased or up-regulated panD, BAPAT, and/orHPDH activity has increased panD, BAPAT, and/or HPDH activity if acomparable fungi having native panD, BAPAT, and/or HPDH activity hasless detectable panD, BAPAT, and/or HPDH activity (for example asmeasured by gene or protein expression).

In one example, a strain of Aspergillus is transformed with a vectorwhich has the effect of up-regulating a panD, BAPAT, and/or HPDH gene(such as a native or non-native panD, BAPAT, and/or HPDH gene). This canbe done by introducing one or more panD, BAPAT, and/or HPDH codingsequences (such as a gene sequence), whose expression is controlled byelements such as promoters and the like which control gene expression,by introducing a nucleic acid sequence which itself (or its encodedprotein) can increase panD, BAPAT, and/or HPDH protein activity in thefungus, or by introducing another molecule (such as a protein orantibody) increases panD, BAPAT, and/or HPDH protein activity in thefungus. For example, a panD, BAPAT, and/or HPDH gene can be up-regulatedby introduction of a vector that includes one or more panD, BAPAT,and/or HPDH sequences (such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 panD,BAPAT, and/or HPDH sequences or copies of such sequences) into thedesired fungus. In some examples, such panD, BAPAT, and/or HPDHsequences are from different fungal species, can be multiple copies froma single species, or combinations thereof, such as panD, BAPAT, and/orHPDH sequences from at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 differentfungal species. In some examples, the panD, BAPAT, and/or HPDHsequence(s) introduced into the fungus is optimized for codon usage.Thus, the disclosure in some examples provides transformed fungi thatinclude at least one exogenous nucleic acid molecule which includes apanD, BAPAT, and/or HPDH gene or coding sequence (such as a nucleic acidsequence encoding SEQ ID NO: 54, 56, or 58, respectively), for examplein combination with ΔcadA. In one example, such transformed cellsproduce more 3HP, for example relative to a comparable fungus with anative cadA.

In one example, the cre-lox system is used for site specificrecombination of DNA (for example see Steiger et al., Appl. Environ.Microbiol. 77(1):114, 2011). Using recombination techniques, thetargeted gene of interest (e.g., cadA) can be deleted in the Aspergillusgenome and replaced with one or more copies of a non-native panD, BAPAT,and/or HPDH sequence (for example in A. terreus, replacing one or bothA. terreus cadA sequences with panD, BAPAT, and/or HPDH sequences fromA. nidulans or A. flavus) flanked by the lox sites. Transient expression(by electroporation of a suicide plasmid containing the cre gene undercontrol of a promoter that functions in Aspergillus) of the crerecombinase should result in efficient elimination of the lox flankedmarker. This process will produce a fungus containing the desiredinsertion mutation and one copy of the lox sequence.

In one example, a transgene is generated and expressed in the desiredfungal cell, such as an ΔcadA fungal cell, to increase panD, BAPAT, andHPDH expression. For example, one or more transgenes can include a panD,BAPAT, and HPDH genomic or cDNA sequence (such as one having at least80%, at least 85%, at least 90%, at least 95%, at least 97%, at least98%, or at least 99% sequence identity to any panD, BAPAT, and HPDHsequence provided herein), for example operably linked to one or morepromoters, such as gpdA and eno1 . In one example, the promoter has atleast 80%, at least 85%, at least 90%, at least 95%, at least 97%, atleast 98%, or at least 99% sequence identity to SEQ ID NO: 74 and/or 77.In some examples, the transgene further includes a trpC transcriptionalterminator sequence of A. nidulans, for example downstream of the panD,BAPAT, and/or HPDH sequence. As an alternative to trpC, othertranscriptional terminators can be used, such as promoters which includea transcriptional terminators (e.g., ArsA7, Arsa-37, polyubiquitin(ubi4)). In one example, the trpC transcriptional terminator has atleast 80%, at least 85%, at least 90%, at least 95%, at least 97%, atleast 98%, or at least 99% sequence identity to SEQ ID NO: 83 or 86. Inone example, the trpC transcriptional terminator comprises or consistsof the sequence shown in SEQ ID NO: 83 or 86. In some examples, thetransgene further includes a ptrA sequence, for example downstream ofthe trpC transcriptional terminator sequence. As an alternative to ptrA,the bleomycin gene or bar gene can be used. In one example, the ptrAsequence has at least 80%, at least 85%, at least 90%, at least 95%, atleast 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:89. In one example, the ptrA sequence comprises or consists of thesequence shown in SEQ ID NO: 89

In one example, the transgene comprises a sequence having at least 80%,at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, orat least 99% sequence identity to SEQ ID NO: 59, 62, 65, 68, 71, and/or74. In one example, the transgene comprises or consists of the sequenceshown in SEQ ID NO: 59, 62, 65, 68, 71, and/or 74.

In one example, the transgene comprises a sequence having at least 80%,at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, orat least 99% sequence identity to SEQ ID NO: 77, 80, and/or 83. In oneexample, the transgene comprises or consists of the sequence shown inSEQ ID NO: 77, 80, and/or 83.

In one example, the transgene comprises a sequence having at least 80%,at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, orat least 99% sequence identity to SEQ ID NO: 86, 89, and/or 92. In oneexample, the transgene comprises or consists of the sequence shown inSEQ ID NO: 86, 89, and/or 92.

In one example, the transgene comprises a sequence having at least 80%,at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, orat least 99% sequence identity to SEQ ID NO: 89 and/or 92. In oneexample, the transgene comprises or consists of the sequence shown inSEQ ID NO: 89 and/or 92.

In one example, the transgene comprises a sequence having at least 80%,at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, orat least 99% sequence identity to SEQ ID NO: 95 and/or 97. In oneexample, the transgene comprises or consists of the sequence shown inSEQ ID NO: 95 and/or 97.

In one example, the transgene comprises a sequence having at least 80%,at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, orat least 99% sequence identity to SEQ ID NO: 59, 62, 65, 68, 71, 74, 77,80, 83, 86, 89, and/or 92. In one example, the transgene comprises orconsists of the sequence shown in SEQ ID NO: 59, 62, 65, 68, 71, 74, 77,80, 83, 86, 89, and/or 92.

In one example, the transgene comprises a sequence having at least 80%,at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, orat least 99% sequence identity to SEQ ID NO: 59, 62, 65, 68, 71, 74, 77,80, 83, 89, and/or 92. In one example, the transgene comprises orconsists of the sequence shown in SEQ ID NO: 59, 62, 65, 68, 71, 74, 77,80, 83, 89, and/or 92.

G. Measuring Gene Expression

A ΔcadA fungus expressing panD, BAPAT, and/or HPDH can be identifiedusing known methods. For example, PCR and nucleic acid hybridizationtechniques, such as Northern, RT-PCR, and Southern analysis, can be usedto confirm that a fungus expresses panD, BAPAT, and/or HPDH such as anincrease in the panD, BAPAT, and/or HPDH copy number.Immunohisto-chemical and biochemical techniques can also be used todetermine if a cell expresses panD, BAPAT, and/or HPDH by detecting theexpression of the panD, BAPAT, and/or HPDH peptide encoded by panD,BAPAT, and/or HPDH. For example, an antibody having specificity forpanD, BAPAT, and/or HPDH can be used to determine whether or not aparticular fungus has increased panD, BAPAT, and/or HPDH proteinexpression, respectively. Further, biochemical techniques can be used todetermine if a cell has increased panD, BAPAT, and/or HPDH expression bydetecting a product produced as a result of the expression of thepeptide. For example, production of 3-HP by ΔcadA A. terreus or A.pseudoterreus can indicate that such a fungus expresses panD, BAPAT, andHPDH.

H. Measuring 3-HP Production

Methods of determining whether a genetic inactivation of cadA incombination with expression of panD, BAPAT, and HPDH in Aspergillusincreases 3-HP production, for example relative to the same strain witha native cadA sequence, (such as a parental strain) include HPLC.

Methods of Producing Aconitic Acid

The recombinant ΔcadA fungi can be used to produce aconitic acid (forexample for as a building block for other materials, such as polymers).Such fungi can be from any Aspergillus species, such as Aspergillusterreus or pseudoterreus. For example, the disclosure provides methodsof making aconitic acid (such as cis-aconitic acid, trans-aconitic acid,or both), which can include culturing ΔcadA fungi under conditions thatpermit the fungus to make aconitic acid, for example in Riscaldatimedium.

In some examples, the fungi are cultured at room temperature (e.g.,20-35° C.) at normal atmospheric pressure (e.g., 1 atm). In someexamples, the method includes purifying or isolating the aconitic acid,for example from the culture media or from the cultured fungus. In someexamples, the aconitic acid is isolated at least 2 days, at least 3days, at least 5 days, at least 8 days or at least 10 days after thestart of culturing, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19 or 20 days after the start of culturing.

Methods of making aconitic acid include culturing ΔcadA fungi underconditions that permit the fungus to make aconitic acid. In general, theculture media and/or culture conditions can be such that the fungi growto an adequate density and produce aconitic acid efficiently. In oneexample the ΔcadA fungi are cultured or grown in an acidic liquidmedium, such as Riscaldati medium (100 g Glucose, 0.11 g KH₂PO₄, 2.36 g(NH₄)₂SO₄, 2.08 g MgSO₄*7H2O, 0.074 g NaCl, 0.13 g CaCl₂*2H₂O, 1 ml of1000× trace elements in 1000 ml DI water, adjust pH to 3.4 with H₂SO₄,1000× trace elements contains 1.3g/L ZnSO₄*7H₂O, 5.5g/L FeSO₄*7H₂O, 0.2g/L CuSO₄*5H₂O, 0.7 g/L MnCl₂*4H₂O). In one example the ΔcadA fungi arecultured or grown in a liquid medium having an initial pH of less than4, such as less than 3.5, for example about pH 3 to 4, 3.5 to 4, 3.3 to3.5, for example pH 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8,3.9 or 4. In some examples the ΔcadA fungi are cultured or grown in aliquid Riscaldati medium at about 20 to 35° C. (such as 20° C. to 30°C., 25° C. to 30° C., 28 to 32° C., or 30° C.) with rotation (such as atleast 100 rpm, at least 120 rpm, such as 150 rpm) at normal pressure.

In one example, the fungi are grown in culture containers (such asbaffled flasks, and in some examples are silanized (5% solution ofdichlorodimethylsilane in heptane (Sigma, St. Louis, Mo.)). Each culturecontainer is inoculated with spores (such as at least 10⁶ spores/ml[agree?]) and incubated for at least 3 days, at least 4 days, at least 5days, or at least 10 days at 30° C. and 100 to 200 rpm to obtainaconitic acid.

In one example, the ΔcadA fungi produce more aconitic acid than acorresponding fungus with wild-type cadA. In specific examples, theΔcadA fungi produce at least 1 g/l of total aconitic acid after 4 days,for example at least 2 g/l, at least 3 g/l, at least 4 g/l, at least 5g/l, at least 6 g/l, at least 7 g/l, at least 8 g/l, at least 9 g/l orat least 10 g/l after at least 5 days, at least 6 days, at least 7 days,at least 8 days, or at least 10 days, such as after 4 to 6 days, 8 to 10days, or 4 to 5 days) when grown in Riscaldati medium at 30° C. with 150rpm shaking. In specific examples, the ΔcadA fungi produce at least 1g/l of cis-aconitic acid after 4 days, for example at least 2 g/l afterat least 5 days, at least 6 days, at least 7 days, at least 8 days, orat least 10 days, such as after 4 to 6 days, 8 to 10 days, or 4 to 5days when grown in Riscaldati medium at 30° C. with 150 rpm shaking. Inspecific examples, the ΔcadA fungi produce at least 1 g/1 oftrans-aconitic acid after 6 days, for example at least 2 g/l, at least 3g/l, at least 4 g/l, at least 5 g/l, at least 6 g/l, at least 7 g/l, atleast 8 g/l, at least 9 g/1 or at least 10 g/l after at least 7 days, atleast 8 days, or at least 10 days, such as after 6 to 12 days, 5 to 10days, or 6 to 10 days) when grown in Riscaldati medium at 30° C. with150 rpm shaking.

In some examples, the method further includes isolating the aconiticacid made by the ΔcadA fungi. Once produced, any method can be used toisolate the aconitic acid. For example, separation techniques (such asfiltration) can be used to remove the fungal biomass from the culturemedium, and isolation procedures (e.g., filtration, distillation,precipitation, electrodialysis, and ion-exchange procedures) can be usedto obtain the aconitic acid from the broth (such as a fungi-free broth).In addition, the aconitic acid can be isolated from the culture mediumafter the aconitic acid production phase has been terminated.

Methods of Producing 3-HP

The recombinant ΔcadA fungi that also express panD, BAPAT, and HPDH canbe used to produce 3-HP

(for example for as a building block for other materials, such asacrylonitrile, acrylic acid by dehydration, malonic acid by oxidation,esters by esterification reactions with alcohols, and reduction to 1,3propanediol). Such fungi can be from any Aspergillus species, such asAspergillus terreus or pseudoterreus. For example, the disclosureprovides methods of making 3-HP, which can include culturing ΔcadA fungithat also express panD, BAPAT, and HPDH under conditions that permit thefungus to make 3-HP, for example in Riscaldati medium (such as modifiedRiscaldati medium with 20× trace elements).

In some examples, the ΔcadA fungi that also express panD, BAPAT, andHPDH are cultured at room temperature (e.g., 20-35° C.) at normalatmospheric pressure (e.g., 1 atm). In some examples, the methodincludes purifying or isolating the 3-HP, for example from the culturemedia or from the cultured fungus. In some examples, the 3-HP isisolated at least 2 days, at least 3 days, at least 5 days, at least 7days, at least 8 days or at least 10 days after the start of culturing,such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19or 20 days after the start of culturing.

Methods of making 3-HP include culturing ΔcadA fungi that also expresspanD, BAPAT, and HPDH under conditions that permit the fungus to make3-HP. In general, the culture media and/or culture conditions can besuch that the fungi grow to an adequate density and produce 3-HPefficiently. In one example the ΔcadA fungi that also express panD,BAPAT, and HPDH are cultured or grown in an acidic liquid medium, suchas Riscaldati medium (100 g Glucose, 0.11 g KH₂PO₄, 2.36 g (NH₄)₂SO₄,2.08g MgSO₄*7H₂O, 0.074g NaCl, 0.13g CaCl₂*2H₂O, 1 ml of 1000× traceelements in 1000 ml DI water, adjust pH to 3.4 with H₂SO₄, 1000× traceelements contains 1.3 g/L ZnSO₄*7H₂O, 5.5 g/L FeSO₄*7H₂O, 0.2 g/LCuSO₄*5H₂O, 0.7 g/L MnCl₂*4H₂O, which may include 20× trace elements).In one example the ΔcadA fungi are cultured or grown in a liquid mediumhaving an initial pH of less than 4, such as less than 3.5, for exampleabout pH 3 to 4, 3.5 to 4, 3.3 to 3.5, for example pH 2.8, 2.9, 3, 3.1,3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9 or 4. In some examples the ΔcadAfungi that also express panD, BAPAT, and HPDH are cultured or grown in aliquid modified Riscaldati medium with 20× trace elements at about 20 to35° C. (such as 20° C. to 30° C., 25° C. to 30° C., 28 to 32° C., or 30°C.) with rotation (such as at least 100 rpm, at least 120 rpm, such as150 or 200 rpm) at normal pressure.

In one example, the fungi are grown in culture containers (such asbaffled flasks, and in some examples are silanized (5% solution ofdichlorodimethylsilane in heptane (Sigma, St. Louis, Mo.)). Each culturecontainer is inoculated with spores (such as at least 10⁶ spores/ml) andincubated for at least 3 days, at least 4 days, at least 5 days, or atleast 10 days at 30° C. and 100 to 300 rpm (such as 150 or 200 rpm) toobtain 3-HP.

In one example, the cadA fungi that also express panD, BAPAT, and HPDHproduce more 3-HP than a corresponding fungus with wild-type cadA(either with or without panD, BAPAT, and HPDH expression). In specificexamples, the ΔcadA fungi that also express panD, BAPAT, and HPDHproduce at least 0.1 g/l of 3-HP after at least 4 days, for example atleast 0.2 g/l at least 0.25 g/l at least 0.3 g/1, at least 0.4 g/l, atleast 0.5 g/l at least 0.6 g/l at least 0.7 g/l at least 0.8 g/l, atleast 0.9 g/1, at least 1.1 g/l, at least 1.2 g/l, at least 1.5 g/l, orat least 1.6 g/l after at least 5 days, at least 6 days, at least 7days, at least 8 days, or at least 10 days, such as after 4 to 6 days, 8to 10 days, or 4 to 5 days, when grown in Riscaldati medium (such asmodified Riscaldati medium with 20× trace elements) at 30° C. with 150rpm shaking.

In some examples, the method further includes isolating the 3-HP made bythe ΔcadA fungi. Once produced, any method can be used to isolate the3-HP. For example, separation techniques (such as filtration) can beused to remove the fungal biomass from the culture medium, and isolationprocedures (e.g., filtration, distillation, precipitation,electrodialysis, and ion-exchange procedures) can be used to obtain the3-HP from the broth (such as a fungi-free broth). In addition, the 3-HPcan be isolated from the culture medium after the 3-HP production phasehas been terminated.

Compositions and Kits

Also provided by the present disclosure are compositions that includeisolated ΔcadA fungi (which in some examples also express panD, BAPAT,and HPDH, such as exogenous panD, BAPAT, and HPDH proteins), such as amedium for culturing, storing, or growing the fungus. In some examples,the ΔcadA fungi and ΔcadA fungi which express panD, BAPAT, and HPDH inthe composition are freeze dried or lyophilized.

Also provided by the present disclosure are kits that include isolatedAcadA fungi (which in some examples also express panD, BAPAT, and HPDH,such as exogenous panD, BAPAT, and HPDH proteins), such as a kit thatincludes a medium for culturing, storing, or growing the fungus. In someexamples, the ΔcadA fungi and ΔcadA fungi which express panD, BAPAT, andHPDH in the kit are freeze dried or lyophilized.

Exemplary mediums include that can be in the disclosed compositions andkits include solid medium (such as those containing agar, for examplecomplete medium (CM) or minimal medium (MM)) and liquid media (such as afermentation broth, such as CM, MM, or CAP medium). In one example, thekit or composition includes Riscaldati medium (100 g Glucose, 0.11 gKH₂PO₄, 2.36 g (NH4)₂SO₄, 2.08 g MgSO₄*7H₂O, 0.074 g NaCl, 0.13 gCaCl₂*2H₂O, 1 ml of 1000× trace elements in 1000 ml DI water, adjust pHto 3.4 with H₂SO₄, 1000× trace elements contains 1.3 g/L ZnSO₄*7H₂O, 5.5g/L FeSO₄*7H₂O, 0.2 g/L CuSO₄*5H₂O, 0.7 g/L MnCl₂*4H₂O), for example

Conc. (g/L) Amount Notes Glucose 100 100 g KH₂PO₄ 0.11 0.11 g (NH₄)₂SO₄2.36 2.36 g MgSO₄ * 7H₂O 2.08 2.08 g NaCl 0.074 0.074 g CaCl₂ * 2H2O0.13 0.13 g ZnSO₄ * 7H₂O 0.0013 0.0013 g Use 1000 X soln. FeSO₄ * 7H₂O0.0055 0.0055 g Use 1000 X soln. CuSO₄ * 5H2O 0.0002 0.0002 g Use 1000 Xsoln. MnCl₂ * 4H₂O 0.0007 0.0007 g Use 1000 X soln. DI Water (L) 1 LAutoclave Time 15 min for small flasks 30 min for large flasks 30-60 forfermenter Comments: Adjust to pH = 3.4 with H₂SO₄

In one example, the kit or composition includes a modified Riscaldatimedium with 20× trace elements, for example 20 times of the following

ZnSO₄ * 7H₂O 0.0013 0.0013 g Use 1000 X soln. FeSO₄ * 7H₂O 0.0055 0.0055g Use 1000 X soln. CuSO₄ * 5H2O 0.0002 0.0002 g Use 1000 X soln. MnCl₂ *4H₂O 0.0007 0.0007 g Use 1000 X soln.

EXAMPLE 1 Materials and Methods

This example describes methods used in the experiments described inExamples 2-6 below.

Strains and vector. The parental wild type A. pseudoterreus strain ATCC32359 was from ATCC. The hygromycin phosphotransferase (hph) markercassette was amplified from vector pCB1003.

Growth conditions. All strains were maintained on complete medium (CM)agar and conidia of spore were harvested from cultures grown for fivedays on complete medium (CM) plate (10 g Glucose, 2 g Triptase peptone,1 g yeast extract, 1 g casamino acids, 50 ml 20×NO₃ Salts, 1 ml of1000×Trace elements, 1 ml of 1000×Vitamin stock, in 1000 ml DI water, pHto 6.5), 20× NO₃ Salts contains in g/l, Na₂NO₃, 120; KCL, 10.4 g;MgSO₄.7H₂O, 10.4 g; KH₂PO₄, 30.4 g. 1000× vitamin solution contains inper 100 ml H₂O: Biotin, 0.01 gm; pyridoxinHCL, 0.01 gm; thiamineHCl,0.01 gm; riboflavin, 0.01 gm; paba, 0.01 gm; nicotinic acid, 0.01 gm,filtered and stock at 4° C. 1000× trace element contains in per 100 mlH₂O: ZnSO₄.7H₂O, 2.2 g; H₃BO₃, 1.1 g; MnCl₂.4H₂O, 0.5 g; FeSO₄.7H₂O, 0.5g; CoCl₂.6H₂O, 0.17 g; CuSO₄.5H₂O, 0.16 g; Na₂MoO₄.2H₂O, 0.15 g;Na₂EDTA, 5 g, add the compounds in order, boil and cool to 60° C. AdjustpH to 6.5 with KOH. Cool to room temperature. Adjust volume to 100 mlwith distilled water.

The transformants were selected for hygromycin resistance on minimummedia (MM) (10 g Glucose, 50 ml 20×NO₃ Salts, 1 ml of 1000×Traceelements, 1 ml of 1000×Vitamin stock, 1000 ml DI water, pH to 6.25˜6.5,hygromycin 100 ug/ml). 0.5×10⁸ conidia were inoculated into 50 ml ofproduction media for itaconic acid production (Riscaldati medium) asdescribed previously (100 g Glucose, 0.11 g KH₂PO₄, 2.36 g (NH₄)₂SO₄,2.08 g MgSO₄.7H₂O, 0.074 g NaCl, 0.13 g CaCl₂.2H₂O, 1 ml of 1000×traceelements in 1000 ml DI water, adjust pH to 3.4 with H₂SO₄, 1000× traceelements contains 1.3 g/L ZnSO₄.7H₂O, 5.5 g/L FeSO₄.7H₂O, 0.2 g/LCuSO₄.5H2O, 0.7 g/L MnCl₂.4H₂O). Cultivation was performed at 30° C. ona rotary shaker at 150rpm. At intervals during the incubation period,three single flasks were harvested for HPLC analysis, biomassmeasurement and RNA extraction. All experiments were replicated threetimes, and the standard deviation of the itaconic acid concentrations ordry weight was always less than 10% of the mean. For collecting samplesfor EST analysis, A. pseudoterreus was grown in 20 liter stirred tankbioreactor.

Construction of deletion mutants. The deletion mutants were constructedby homologous double crossover with fusion PCR products. Syntheticoligos used for each construct are described in Table 1.Oligonucleotides were from IDT (Coraville, Iowa). Ex Taq polymerase(TaKaRa, Japan) was used to generate DNA constructs for making geneknockouts. Briefly, the 5′ flanking region (˜1.5 kb) of the target genewas amplified by primer pair F1 and R3. The 3′ flanking region (˜1.5 kb)of the target genes was amplified by primer pair F4 and R6. R3 and F4carried 20-25 bases complementary to 5′ and 3′ ends of the hph cassette,respectively. The hph marker cassette was amplified from pCB1003 withthe hphF and hphR primers that carried 30 bases complementary to the 3′end of the 5′ flanking region and the 5′ of the 3′ flanking region,respectively. The three fragments, including the 5′ flanking region, thehph marker cassette and the 3′ flanking region were mixed in 1:3:1 molarratio and combined by overlap PCR during the second round PCR. In thethird round of PCR, the fusion PCR product was amplified with a nestedprimer pair (F2 and R5). This final PCR product carried a hygromycinmarker cassette flanked by sequences homologous to the upstream anddownstream regions of the target gene. 1-2 ug of the final product wasused to transform strain A. pseudoterreus strain ATCC 32359.

TABLE 1 Primers for making deletion constructs gene targeted primer nameprimer sequence (SEQ ID NO) tf at tff1 gagccatagccatgcaagcg (1) at tff2atagagtccttggatgagacg (2) at tfr5 gtggatttcgaggttccttgc (3) at tfr6gaagtagaaccatgtggatcg (4) at hphf tfr3tgacctccactagctccagcactactagataggcccgtttagagagtgcc (5) at hphr tff4aatagagtagatgccgaccggccgcttcgacgacagctctgcactctcc (6) at tfr3hphfggcactctctaaacgggcctatctagtagtgctggagctagtggaggtca (7) at tftff4hphrggagagtgcagagctgtcgtcgaagcggccggtcggcatctactctatt (8) mttA at motf1gctgcatactcggattacgc (9) at motf2 Gaaaaggtactcggagtacg (10) at motr5cagaccaaggagctttcctg (11) at motr6 cattaagccacaggcttgcg (12) athphfmotr3tgacctccactagctccagcaatatggatgctgttcgttcgccgtgctgg (13) athphrmotf4aatagagtagatgccgaccgtgacgaggatgtgctgagtccaaacaaagc (14) at motr3hphfccagcacggcgaacgaacagcatccatattgctggagctagtggaggtca (15) at motf4hphrgctttgtttggactcagcacatcctcgtcacggtcggcatctactctatt (16) cadA at cadf1ctccagtaacagaaccgacc (17) at cadf2 gaacttcactgccgcattgg (18) at cadr5ggacactccaagaggataagg (19) at cadr6 gctcatcacattgtttgccg (20)at hphfcadr3 tgacctccactagctccagcggtcaatttaagaggacgatcttcgctgcg (21)at hphrcadf4 Aatagagtagatgccgaccgtcagcctggacaggctcaccgacattagcc (22)at cadr3hphf cgcagcgaagatcgtcctcttaaattgaccgctggagctagtggaggtca (23)at cadf4hphr ggctaatgtcggtgagcctgtccaggctgacggtcggcatctactctatt (24)mfsA mfsf1 tgatgagctgaattcgttgc (25) mfsf2 tatagccagcttttgctgtg (26)mfsr5 catagcgttcagagtgttg (27) mfsr6 ccatttcaatgctttgtgcg (28) mfsr3hphfccataccacccttaccctcttggagtgtccgctggagctagtggaggtca (29) mfsf4hphrgctgtggcctcctggcgattacgcaatattcggtcggcatctactctatt (30) hphfmfsr3tgacctccactagctccagcggacactccaagagggtaagggtggtatgg (31) hphrmfsf4aatagagtagatgccgaccgaatattgcgtaatcgccaggaggccacagc (32) p450 p450f1tctccaaatcatcgtcatcg (33) p450f2 cttcaatcgcaccgacatcc (34) p450r5tcgtgtagacaagtccagtc (35) p450r6 ctataccactctagtgatgg (36) p450r3hphfcctctgctcaggttgttttcgaacaggagcgctggagctagtggaggtca (37) p450f4hphrcggaatgcagataggcatcacagtccagaacggtcggcatctactctatt (38) hphfp450r3tgacctccactagctccagcgctcctgttcgaaaacaacctgagcagagg (39) hphrp450f4aatagagtagatgccgaccgttctggactgtgatgcctatctgcattccg (40)

Transformation of A. pseudoterreus protoplasts. 10⁸ conidia of A.pseudoterreus ATCC 32359 were used to inoculate 300 ml Erlenmeyer baffleflasks containing 100 ml of complete media. The cultures were grownovernight (16-18 hrs) at 30° C. and 150 rpm. The mycelia were thenharvested by filtering the culture through miracloth and rinsing themycelia mat with sterile water. The protoplasts were prepared bytreating mycelia (mass of approximately 1-2 beans) with 20 mg/ml lysingenzyme (L1412, Sigma) dissolved in 20 ml of osmotic wash buffer (0.5MKCl, 10 mM sodium phosphate, pH 5.8) for 2 h. Protoplasts were collectedby filtering protoplasts through sterile miracloth into a 50 ml screwcap centrifuge tube and centrifuging at 1000×g for 10 min at 4° C.Protoplasts were then washed twice with 20 ml Washing Solution (0.6MKCl, 0.1M Tris/HCl, pH 7.0) and once in 10 ml Conditioning Solution(0.6M KCl, 50 mM CaCl₂, 10 mM Tris/HCl, pH 7.5). For transformation, 1-2ug DNA was added to 2×10⁷ protoplasts in 0.1 ml Conditioning Solution. Acontrol reaction without added DNA was performed at the same time. 25 μlof PEG solution (25% PEG8000, 0.6M KCl, 50 mM CaCl₂, 10 mM Tris/HCl, andpH 7.5) was added and the protoplasts were incubated for 20 min on ice.An additional 500 ul of the PEG was added using a wide bore pipette tipand carefully mixed with the protoplasts by gently pipetting up and down1-2 times. The protoplast solution was then incubated for 5 min on ice.1 ml of cold Conditioning Solution was added and mixed by gentlyinverting the tube several times. Then the protoplast suspension wasmixed with 12 ml of 50° C. selection agar (Minimal media+0.6M KCl+1.5%Agar+100 ug/ml hygromycin) contained in a 15 ml screwcap centrifugetube. The tubes were then mixed by inverting the tubes 3-4 times andpoured directly onto the petri dish plates. The control reaction wasdivided into a positive control plate (no selection antibiotics in thetop agar and bottom plates) and a negative control (with selectionhygromycin in top and bottom agar). The solidified plates were invertedand incubated overnight at 30° C. The next day, the plates were thenoverlaid with 15 ml of Minimal Medium (MM) containing 150 ug/mlhygromycin. Colonies should start to appear after 3-4 days. Thetransformants were excised and transferred to MM plate containing 100ug/ml hygromycin. Correct transformants on the hygromycin plate wereconfirmed by PCR approaches and southern blot.

Dry mass measurement. Dry mass at each time point was determined byharvesting the mycelium on miracloth by suction filtration and washedtwice with 50 ml distilled water. Subsequently, the dry weight wasdetermined by drying it overnight in pre-weighed tubes on lyophilizer.

HPLC. The content of itaconic acid, aconitic acid, and glucose in eachsample collected from filtration (0.22 um) was assayed by ahigh-pressure liquid chromatography (HPLC) on a Bio-Rad Aminex HPX-87Hion exclusion column (300 mm×7.8 mm). Columns were eluted with Sulfuricacid (0.005 M) at a flow rate of 0.55 mL/min. The sample volume was10-100 ul, and IA was detected at 210 nm with a Waters 2414 refractiveindex detector.

RNA isolation and transcript analysis by Quantitative real time RT-PCR.Wild type and tf deletion strains were grown in Riscaldati medium at 30°C. After 3 days growth, mycelia were harvested, pressed dry betweenpaper towels and immediately flash frozen in liquid nitrogen. The entiresample was then ground in a mortar and pestle with liquid nitrogen.Approximately 100 mg samples (about 0.1 ml) were extracted using Trizol®reagent (Chomczynski, BioTechniques 1993, 15(3):532-534, 536-537) andthe resulting RNA was converted to cDNA using high capacity RNA-to-DNAkit (Applied Biosystems). Quantitative RT-PCR were performed in 50 ulreactions containing 25 ul of Power SYBR green PCR master mix (AppliedBiosystems), 50 ng cDNA (from 50 ng RNA) and 0.2 uM forward and reverseprimers. The RT-PCR primers used for analysis of the mttA, cadA, mfsAgenes and benA (β-tubulin) as endogenous control gene are listed inTable 3. There are two additional controls, one is a no RT (withoutadding RT enzyme mix) control to estimate contamination from genomicDNA, and the other is no-template controls for each primer pair tomeasure effect from primer dimer formation . Amplification was performedusing 7900HT Fast Real-Time PCR system (Applied Biosystems) programmedto initially hold at 95° C. for 10 min and then to complete 45 cycles of95° C. for 15 s, 60° C., for 60 s. The data were analyzed using thecomparative C_(T) method (e.g., see Schmittgen et al., Analyticalbiochemistry 2000, 285(2):194-204)

EXAMPLE 2 Expression Profile of Itaconic Acid Gene Cluster in A.pseudoterreus

RNA samples were prepared from three different growth stages of A.pseudoterreus in the itaconic acid production process. The stageswere 1) “pre-production,” before itaconic acid production begins, 2)“production onset,” the beginning of itaconic acid production correlatedwith phosphate depletion, and 3) “production,” early in the phase ofmaximum itaconic acid production rate (FIG. 2). EST data revealed fourgenes in the cluster having high expression frequency both in the onsetphase and production phase, but not in the pre-production phase (Table2). These genes were tf, mttA, cadA, and mfsA.

TABLE 2 Number of ESTs per gene at three stages of itaconic acidproduction Broad Institute Gene Pre- Production Gene No. DescriptionProduction Onset Production ATEG_09968.1 upstream 0 0 0 flanking gene;lovE ATEG_09969.1 tf 0 4 4 ATEG_09970.1 mttA 0 81 93 ATEG_09971.1 cadA 077 110 ATEG_09972.1 mfsA 0 6 7 ATEG_09973.1 p450 0 7 11 ATEG_09974.1downstream 0 0 0 flanking gene ATEG_09817.1 control; gapdh 31 51 43

cadA has 77 ESTs at the beginning of itaconic acid (IA) production and110 ESTs during the IA production, while mttA has 81 and 93 ESTsrespectively in each stage. Both have no transcript detected before IAis produced. Transcription factor (tf) and mfsA, like cadA and mttA, didnot show any expression before IA production, but had significant levelsof transcription following the initiation of itaconic acid production(Table 2).

When genes upstream and downstream of tf, cadA, mttA and mfsA wereexamined, a similar expression pattern was not observed. No transcriptwas detected for either upstream or downstream genes in any stage of IAproduction except for p450. Control gene gpdh, which is far away fromthis region, showed high expression through the whole growth stage. ThisEST data clearly demonstrated that four genes tf, cadA, mttA and mfsAhave the same expression pattern and are closely related to the IAproduction process. In addition, these four genes are in the samecluster. They are turned on strongly at the onset of IA production andpersists through the production phase (FIG. 2).

EXAMPLE 3 Effect of tf, cadA, mttA and mfsA Deletion on Itaconic AcidProduction in A. pseudoterreus

A transformation system was developed to allow for transformation of A.pseudoterreus (see Example 1). This system was used to generaterecombinant knockout strains for each of the endogenous tf, cadA, mttAand mfsA genes. The KO mutant strains were confirmed by PCR and southernblot. The transformation protocol gave very high frequency of homologousdeletion, 8 out 10 had the correct deletion. This high deletionfrequency may be due to the presence of a ku gene mutation in the genomeof wild-type A. pseudoterreus.

Biomass accumulation and itaconic acid (IA) production of each of thefour knockout mutants and wild type A. pseudoterreus were measured atday 5. All strains, including wild type, had similar biomassaccumulation (FIG. 3A). There is no significant difference in biomassamong these five strains, indicating that deletion of these genes doesnot cause a noticeable growth defect.

However, the yield of IA was significantly lower in all four deletionstrains (Δtf, ΔcadA, ΔmttA and ΔmfsAΔ) when compared to wild type A.pseudoterreus. After 5 days growth in the Riscardati medium, the Δtfstrain had only generated ˜3g/l IA, compared to the wild type strain,which generated ˜24 g/l of IA (about an 8-fold decrease). No detectableIA was produced by the ΔcadA and ΔmttA strains. ΔmfsA produced around 16g/L itaconic acid, about ⅔ of wild type A. pseudoterreus.

These observations demonstrate that tf, mttA, cadA and mfsA genes play arole in itaconic acid production.

EXAMPLE 4 Production Kinetics of Itaconic Acid in Wild Type and tfDeletion Strain

To test the production kinetics in the deletion strains, Δtf and wildtype A. pseudoterreus strain ATCC 32359 were tested for IA productionduring the growth on a rotary shaker for 7 days. IA was analyzed by HPLCfor 2, 4, 6 and 7 day cultures.

As shown in FIG. 4, the IA yield plateaued at day 7 in both Δtf and wildtype strains. Interestingly, the IA yield in Δtf (5g/l) is much lowerthan that of wild type (35 g/l), a decrease of about 7-fold. Thus, theΔtf strain produces IA at slower rate with a lower maximum IA yield thanthe wild type strain.

EXAMPLE 5 tf Regulation

The effects of tf gene deletion on the transcription level of othergenes in the cluster were investigated by real-time reversetranscription PCR (RT-PCR). In the both Δtf and wild type strains,expression level of each gene was analyzed by RT-PCR by measuring mttA,cadA, mfsA mRNA levels using primers specific for those genes (Table 3).

TABLE 3 primers for real-time RT-PCR analysis of clustergene transcript level Gene targeted Primer namePrimer sequence (SEQ ID NO:) mttA mttF Gctttcaatgtggttcctac (41) mttRctccatcacctaccctttc (42) cadA cadF gaagtgtgggatctggc (43) cadRgggttcggtatttgtgaag (44) mfsA mfsF caagaacagtttggcctgag (45) mfsRgcggacatcatacaatctgg (46) benA β -tubulinF ttgtcgatgttgttcgtcgc (47)β-tubulinR tggcgttgtaaggctcaacc (48)

As shown in FIG. 5, in Δtf strains, mRNA level of mttA decreased 57fold, cadA mRNA level decreased 37 fold, and mfsA decreased 23 fold, ascompared to their expression in wild type A. pseudoterreus 32359. Thus,inactivation of the tf gene dramatically reduced the level of mRNA ofother genes in the cluster. Within the itaconic acid biosynthesiscluster, the transcription factor potentially controls expression ofother genes.

EXAMPLE 6 cadA Deletion Creates a Novel Strain that Produces AconiticAcid

In A. pseudoterreus, when cadA was deleted, itaconic acid production wascompletely abolished (FIG. 3B). However, 3.5 g per liter aconitic acidin the ΔcadA strain was detected at day 5 (FIG. 6A). Aconitic acid wasnot produced by the wild type, ΔmttA or ΔmfsA strains (FIG. 6A). A timecourse analysis showed that aconitic acid started to appear in thesupernatant at day 3, similar as IA in the wild type strain (FIG. 6B).At day 3, only cis-aconitic acid was detected in the supernatant. At day4, both cis-aconitic acid and trans-aconitic acid were detected. Fromday 5 onward, cis-aconitic acid remained consistent at about 2 g/L,while trans-aconitic acid yield continued to increase (FIG. 6B). By day10, 10 g/L trans-aconitic acid was detected in the supernatant from theΔcadA strain (FIG. 6B). FIG. 6C shows a comparison of total aconiticacid production between wild type and ΔcadA fungi. Thus, ΔcadA stains ofA. pseudoterreus and A. terreus can be used to produce cis- andtrans-aconitic acid.

EXAMPLE 7 Materials and Methods

This example describes methods used in the experiments described inExample 8.

Transgene Expression Vector for 3-HP Production

Isolation of DNA Fragments:

Fragment 1: A. pseudoterreus 5′-cadA gene, 987 bp (SEQ ID NO: 59)isolated by PCR with the oligo pair 1969 and 1970 (SEQ ID NOS: 60 and61, respectively) and A. pseudoterreus genomic DNA;

Fragment 2: A. niger gpdA promoter, 813 bp (SEQ ID NO: 62) isolated byPCR with oligo pair of 1971 and 1972 (SEQ ID NOS: 63 and 64,respectively) and A. niger genomic DNA;

Fragment 3: aspartate 1-decarboxylase (panD) cDNA of Tribolium castaneumwith codon optimization for A. pseudoterreus, 1617 bp (SEQ ID NO: 65)was isolated by PCR with the oligo pair of 1973 and 1974 (SEQ ID NOS: 66and 67, respectively) and the plasmid DNA containing the synthesizedpanD cDNA;

Fragment 4: bidirectional terminator from A. niger elf3/multifunctionalchaperone (SEQ ID NO: 68) was isolated by PCR with oligo pair of 1975and 1976 (SEQ ID NOS: 69 and 70, respectively) and the genomic DNA of A.niger;

Fragment 5: codon optimized synthetic cDNA of β-alanine-pyruvateaminotransferase (BAPAT) of Bacillus cereus, 1350 bp (SEQ ID NO: 71) wasisolated by PCR with oligo pair of 1977 and 1978 (SEQ ID NOS: 72 and 73,respectively) and the plasmid DNA containing the synthesized BABAT cDNA;

Fragment 6: A. niger eno1 promoter, 704 bp (SEQ ID NO: 74) isolated byPCR with oligo pair of 1979 and 1980 (SEQ ID NOS: 75 and 76,respectively) and A. niger genomic DNA;

Fragment 7: A. nidulans gpdA promoter, 885 bp (SEQ ID NO: 77) wasisolated by PCR with the oligo pair of 2002 and 1982 (SEQ ID NOS: 78 and79, respectively) and A. nidulans genomic DNA;

Fragment 8: the codon optimized synthetic cDNA of E. coli3-hydroxypropionate dehydrogenase (HPDH), 741 bp (SEQ ID NO: 80) wasisolated by PCR with oligo pair of 1983 and 1984 (SEQ ID NOS: 81 and 82,respectively) and the plasmid DNA containing the codon-optimizedsynthesized HPDH DNA of E. coli;

Fragment 9: trpC terminator of A. nidulans, 473 bp (SEQ ID NO: 83)isolated by PCR with oligo pair of 1985 and 2004 (SEQ ID NOS: 84 and 85,respectively) and plasmid DNA of pAN7.1;

Fragment 10: trpC terminator of A. nidulans, 473 bp (SEQ ID NO: 86)isolated by PCR with the oligo pair of 2005 and 1986 (SEQ ID NOS: 87 and88, respectively) and plasmid DNA of pAN7.1;

Fragment 11: A. oryzae ptrA selection marker gene, 2005 bp; SEQ ID NO:89) isolated by PCR with the oligo pair of 1987 and 1988 (SEQ ID NOS: 90and 91, respectively) and A. oryzae genomic DNA;

Fragment 12: A. pseudoterreus 3′-cadA gene, 908 bp (SEQ ID NO: 92)isolated by PCR with the oligo pair 1989 and 2003 (SEQ ID NOS: 93 and94, respectively) and A. oryzae genomic DNA;

Fragment 13 (SEQ ID NO: 95): Combination of Fragments 7 to 9 (SEQ IDNOS: 77, 80, and 83, respectively), 2099 bp isolated by PCR with oligopair of 1981 and 1986 (SEQ ID NOS: 96 and 88, respectively) and plasmidDNA of pZD-2; and

Fragment 14 (SEQ ID NO: 97): Combination of Fragments 11 to 12 (SEQ IDNOS: 89 and 92, respectively), 2913 bp was isolated by PCR with theoligo pair of 1987 and 1990 (SEQ ID NOS: 90 and 98, respectively) andplasmid DNA of pZD-3.

The oligonucleotide primers used are shown in Table 4.

TABLE 4 Primers used to generate vector for 3-HP production NameSequence (SEQ ID NO:) 1969cad1 ccctcgaggtcgacggtatcgata GATATCGGTTGTAGCAGCGTAAA CAC (60) 1970cad2tctttcatagtagCCTTGGTGAACATCTTGAGG (61) 1971gpdA1atgttcaccaaggCTACTATGAAAGACCGCGATG (63) 1972gpdA2cgccggtggcgggCATTGTTTAGATGTGTCTATGTG (64) 1973pan1catctaaacaatgCCCGCCACCGGCGAGGACCA (66) 1974pan2atccaacccatcaGAGGTCGGAGCCCAGGCGTTCG (67) 1975ter1gggctccgacctcTGATGGGTTGGATGACGATG (69) 1976ter2tctggcccagctcTGAGTCCTAGATGGGTGGTG (70) 1977bap1catctaggactcaGAGCTGGGCCAGACATTCCTTC (72) 1978bap2gtccatcaacatgGAACTGATGATCGTCCAGGTCAC (73) 1979eno1cgatcatcagttcCATGTTGATGGACTGGAGGG (75) 1980eno2gaactagtggatcccccgggctgcGttaaCTCGAGCTTACAAGAAGTA GCC (76) 1981gpdA1acaggctacttcttgtaagctcgagttTCTGTACAGTGACCGGTGAC (96) 1982gpdA2tgaccagcacgatCATGGTGATGTCTGCTCAAG (79) 1983hpd1agacatcaccatgATCGTGCTGGTCACGGGCGC (81) 1984hpd2gccatcggtcctaTTGGCGGTGGACGTTCAGGC (82) 1985trp1cgtccaccgccaaTAGGACCGATGGCTGTGTAG (84) 1986trp2cccgtctgtcagaGAGCGGATTCCTCAGTCTCG (88) 1987ptrA1gaggaatccgctcTCTGACAGACGGGCAATTGATTAC (90) 1988ptrA2gaatgttgctgagGAGCCGCTCTTGCATCTTTG (91) 1989cad3gcaagagcggctcCTCAGCAACATTCGCCATGTTC (93) 1990cad4actaaagggaacaaaagctggagctCAGCTCCACTGCTCATAGTCTTT G(98) 2002gpdA5ccctcgaggtcgacggtatcgataGTTAACTCTGTACAGTGACCGGTG AC (78) 2003cad3gaactagtggatcccccgggctgcaCAGCTCCACTGCTCATAGTCTTT G (94) 2004trpRgaactagtggatcccccgggctgcaGAGCGGATTCCTCAGTCTCG (85) 2005trpFccctcgaggtcgacggtatcgataTAGGACCGATGGCTGTGTAG (87)

An overview of the arrangement of the Fragments is shown in FIG. 7.Fragments 1 to 6 (SEQ ID NOS: 59, 62, 65, 68, 71 and 74, respectively)were assembled into the plasmid DNA pBlueScript SK (−) linearized withHindIll and Pstl via Gibson Assembly master kit to form plasmid pZD-1. Arestriction enzyme site HpaI was introduced at the end of the fragment 6for further cloning.

Fragments 7 to 9 (SEQ ID NOS: 77, 80, and 83, respectively) wereassembled into the plasmid DNA pBlueScript SK (−) linearized withHindIII and PstI via Gibson Assembly master kit to form plasmid DNApZD-2.

Fragments 10 to 12 (SEQ ID NOS: 86, 89, and 92, respectively) wereassembled into the pBlueScript SK(−) vector linearized with restrictionenzyme HindIII and PstI by Gibson assembly to form the plasmid vectorZD-3. (Only fragments 11 and 12 were used in the next step; SEQ ID NOS:89 and 92).

Fragments 13 and 14 (SEQ ID NOS: 95 and 97) were assembled together intothe plasmid DNA vector ZD-1 linearized with restriction enzyme HpaI/SacIvia Gibson Assembly master kit to form pZD-4.

Genomic DNA isolation and Southern blotting analysis were performed asdescribed in Example 1 (and see Dai et al., 2017, Appl MicrobiolBiotechnol 101:6099-6110).

Detection of 3-HP

The extracellular 3-HP in the culture supernatants was quantified withHPLC method as described in Example 1.

EXAMPLE 8 Production of 3-HP

The constructs generated in Example 7 (FIG. 7) were transformed intowild type A. pseudoterreus strain ATCC 32359 using he methods describein Example 1, thereby inactivating/disrupting the cadA gene in someexamples.

As shown in FIG. 8, restriction fragment length polymorphism of selectedtransgenic strains show that the transgene expression cassette wasinserted into the cadA locus in strain-2 (with one copy) and strain-6(two copies), while the strain-4 and strain-5 carry the transgeneexpression cassette with random integration. No integration of transgeneexpression cassette was observed in strain-1 and strain-3.

3-HP production was measured in several transformants. As shown in FIG.9A, the ΔcadA strain did not produce 3-HP, while insertion of thetransgene expression cassette that allowed for expression of panD,BAPAT, and HPDH, into the cadA locus with one copy or two copies andresulted in 0.9 or 1.7 g/l 3-HP accumulation in the strains 3HP-2 or3HP-6. In contrast, when the transgene expression cassette was randomlyinserted into the chromosome, 3HP production was substantially lower(Strains 3HP-4 and 3HP-5). FIG. 9B shows 3-HP production over 8 days inStrains 3HP-2 and 3HP-6 (strains 2 and 6, respectively). Thus,genetically inactivating cadA can increase 3-HP production.

In view of the many possible embodiments to which the principles of thedisclosure may be applied, it should be recognized that the illustratedembodiments are only examples of the invention and should not be takenas limiting the scope of the invention. Rather, the scope of theinvention is defined by the following claims. We therefore claim as ourinvention all that comes within the scope and spirit of these claims.

We claim:
 1. An isolated recombinant Aspergillus fungus comprising agenetic inactivation of an endogenous cis-aconitic acid decarboxylase(cadA) gene.
 2. The isolated recombinant Aspergillus fungus of claim 1,wherein the Aspergillus fungus is Aspergillus pseudoterreus.
 3. Theisolated recombinant Aspergillus fungus of claim 1, wherein theAspergillus fungus is Aspergillus terreus.
 4. The isolated recombinantAspergillus fungus of claim 1, wherein the endogenous cadA gene isgenetically inactivated by complete or partial deletion mutation or byinsertional mutation.
 5. The isolated recombinant Aspergillus fungus ofclaim 1, wherein the cadA gene prior to its genetic inactivation encodesa protein having at least 80% sequence identity to SEQ ID NO: 50 or 52.6. The isolated recombinant Aspergillus fungus of claim 1, wherein thecadA gene prior to its genetic inactivation comprises a coding sequencecomprising at least 80% sequence identity to SEQ ID NO: 49, 51, 59 or92.
 7. A composition comprising the isolated recombinant Aspergillusfungus of claim
 1. 8. A kit, comprising: the isolated recombinantAspergillus fungus of claim 1; and a medium for culturing the fungus. 9.A method of making aconitic acid, comprising: culturing the isolatedrecombinant Aspergillus fungus of claim 1 under conditions that permitthe fungus to make aconitic acid; thereby making aconitic acid.
 10. Themethod of claim 9, wherein the fungus is cultured in Riscaldati medium.11. The method of claim 9, further comprising isolating the aconiticacid from culture media or from the fungus.
 12. The method of claim 9,wherein the aconitic acid is cis-aconitic acid.